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Biomedical subjects

A Goldfarb

Publications and source records attributed to A Goldfarb.

At least 109 records · Page 6Linked to original sources
PubMed1988

Late sigma factor of bacteriophage T4. Formation and properties of RNA polymerase-promoter complexes.

Bacteriophage T4 late gene promoters do not display sequence homology in the -35 region (Christensen, A. C., and Young, E. T. (1982) Nature 299, 369-371), suggesting an unusual geometry of RNA polymerase-promoter interaction. We have analyzed in vitro utilization of a late T4 promoter by RNA polymerase reconstituted from E. coli core enzyme (E) and bacteriophage T4 late sigma factor (sigma gp55). The E sigma gp55 holoenzyme forms a stable promoter complex which lacks protein-DNA contacts upstream from position -30 and is sensitive to direct attack by heparin. This complex is capable of reiterative oligonucleotide synthesis (abortive initiation). Kinetic analysis of complex formation reveals a rapidly forming inactive intermediate (closed complex) which is slowly isomerized into a catalytically active form (open complex). The results indicate that all components essential for promoter binding, open complex formation, and initiation of transcription are present in the "downstream" part of the RNA polymerase molecule, which is defined by the -30 to +20 footprint. On the basis of these observations and the results of others, we suggest that during transcription initiation at bipartite (Escherichia coli) promoters, an "upstream" DNA-binding domain of RNA polymerase which recognizes specific sequence elements in the -35 region plays an auxiliary role by regulating the rate of productive interactions in the downstream part of the molecule through an allosteric mechanism.

Base Sequence↗
PubMed1988

Enhanced pirmenol elimination by rifampin.

The potential for drug-drug interaction between pirmenol, an extensively metabolized antiarrhythmic agent, and rifampin, a potent inducer of hepatic drug-metabolizing enzymes, was evaluated in 12 healthy adults. After administration of a single 150-mg oral dose of pirmenol on day 1, pirmenol plasma and urine concentrations were determined for 72 hours postdose. On days 4 through 17, subjects received 600 mg of rifampin once daily. On day 15, subjects were given a second single 150-mg oral dose of pirmenol concomitantly with rifampin, and plasma and urine concentrations were again determined. Coadministration of rifampin with pirmenol resulted in significant (P less than .005) changes in pirmenol pharmacokinetic parameters. A sixfold decrease in pirmenol AUC and sevenfold increase in the apparent plasma clearance of pirmenol were found. Elimination half-life decreased more than twofold. Based on these findings, pirmenol dosage adjustment will be required when pirmenol is given to patients concurrently receiving rifampin. These results suggest that the administration of pirmenol with other agents that induce hepatic enzymes may result in accelerated pirmenol clearance.

Adult↗
PubMed1988

Detection of specific beta-globin mutations in Kurdish Jews with beta-thalassemia.

Patients with beta-thalassemia, of Kurdish extraction, were screened for the presence of two mutations, in the TATA box and in codon 44, previously discovered in this ethnic isolate. Of the 56 chromosomes analyzed, 13 were found to carry the TATA box mutation and 17 the codon 44 mutation. The result of this work provides a basis for a more efficient prenatal diagnosis program for this community.

Adolescent↗
PubMed1987

Competition between sigma factors for core RNA polymerase.

The switch of RNA polymerase specificity from early to late promoters of bacteriophage T4 is achieved by substitution of host sigma factor, sigma 70, with the T4 induced factor, sigma gp55. However, overproduction of sigma gp55 from an expression vector is not detrimental to Escherichia coli growth. Direct competition binding assays demonstrate that sigma 70 readily displaces sigma gp55 from RNA polymerase and thereby reverses the promoter specificity of the enzyme. The displacement also occurs with the core enzyme modified by bacteriophage T4 infection. We postulate that an antagonist of sigma 70 should be formed in T4-infected cells to aid sigma gp55 in the early/late switch.

Binding, Competitive↗
PubMed1987

Overexpression and purification of a biologically active rifampicin-resistant beta subunit of Escherichia coli RNA polymerase.

The gene rpoB (rifD 18), which encodes rifampicin-resistant beta subunit of Escherichia coli RNA polymerase, has been placed on an overexpression plasmid under the control of bacteriophage T7 promoter. Induction of the T7 RNA polymerase gene in the host cells resulted in extensive overproduction of the beta polypeptide. Most of the overproduced material was recovered from cell lysates in insoluble form and was solubilized by extraction with 6 M urea. Purified overproduced beta subunit was added, in molar excess, to urea-denatured rifampicin-sensitive RNA polymerase. Upon removal of urea by dialysis, the reconstituted enzyme became rifampicin-resistant, indicating that overproduced beta subunit can be efficiently assembled into functional holoenzyme.

DNA-Directed RNA Polymerases↗
PubMed1986

A protective human monoclonal IgA antibody produced in vitro: anti-pneumococcal antibody engendered by Epstein-Barr virus-immortalized cell line.

Human lymphocytes that produce anti-pneumococcal antibodies were separated and immortalized by Epstein-Barr virus and then cloned. One clone (NAD-Sel) produces an IgA, kappa antibody which is specific for the polysaccharides of type 8 pneumococcus, while not reactive with any of the polysaccharides derived from 24 other pneumococcal strains. The antibody, which is present in the cell supernatant as monomer and polymer, binds to protein A and does not fix complement. When incubated in vitro with type 8 pneumococci, it induces direct killing and increases the opsonization of these bacteria by mouse macrophages.

Animals↗
PubMed1986

Erythropoietin activity in the serum of beta thalassemic patients.

Serum erythropoietic activity was determined in 32 patients with beta thalassemia major and intermedia. Quantitation was performed by an in vitro bioassay using rabbit erythroid precursor cells (CFU-E) either by colony assay or by 3H-thymidine uptake. 20 polytransfused beta-thalassemic major patients had erythropoietic activity (mean 89.3 +/- 36 milliunits/ml) which was not significantly different (p greater than 0.2) from normal individuals (51.3 +/- 32 milliunits/ml). 12 untransfused patients with beta thalassemia intermedia were found to have comparable serum erythropoietic activity (p greater than 0.01). These levels were much lower than those found in patients with aplastic anemia who had a comparable degree of anemia. We have shown that the low EPO activity in thalassemic patients was not due to experimental conditions (excess of ferritin, low transferrin) nor to specific inhibitors appearing in this disease. No correlation was found between the erythropoietic activity and sex or other clinical parameters of the patients such as severity of the anemia, splenectomy, iron chelation or transfusion therapy. 4 young thalassemic children (1-2 yr of age) studied had high erythropoietic activity ranging from 661 to 5793 milliunits/ml--significantly different from normal children of the same age. It is suggested, therefore, that a decrease in serum erythropoietin levels develops during the course of the disease.

Anemia, Aplastic↗
PubMed1986

Beta-thalassemia: analysis of mRNA precursors of a mutant human globin gene with defective splicing using peripheral blood nucleated red blood cells.

Studies on the effects of thalassemic mutations on gene function in vivo have clinical as well as scientific implications. Usually these studies have been performed on nucleated red blood cell (RBC) precursors normally present in bone marrow. Many patients with beta-thalassemia are splenectomized and may have high levels of nucleated RBC, orthochromatic normoblasts, in their peripheral blood (1-5% of total RBC). The possibility of exploiting these cells instead of bone marrow as a source for nuclear and cytoplasmic RNA for expression studies was investigated. A simple procedure was developed for enrichment for normoblasts in blood samples withdrawn from patients prior to transfusion. Globin transcripts were analyzed in RNA purified from 12 patients. Unspliced precursor beta-mRNA molecules were observed in a patient with beta o-thalassemia, homozygous for a mutation at the 5' IVS2 splice site of the beta-globin gene. Detailed analyses showed that his mature beta-mRNA was larger than normal, and that a cryptic 5' splice site, approximately 50 nucleotides downstream from the normal one, was utilized. We conclude that peripheral blood can be used as a reliable source of RNA for the analysis of the effects of beta-thalassemia mutations on gene expression and the relationship to the clinical condition. Moreover, this procedure facilitates the comparison of in vivo gene expression with the results obtained from DNA transfection experiments with cloned beta-thalassemia genes.

Adolescent↗
PubMed1986

[Familial cardiomyopathy caused by carnitine deficiency].

The authors report a familial case of carnitine insufficiency presenting in two out of seven children as a severe, isolated, hypertrophic and hypokinetic cardiomyopathy. The etiology was confirmed by histological study and measurement of carnitine concentrations in the blood and muscle. The evolution was spectacular with specific therapy. Left ventricular hypokinesia regressed completely within 18 months (fractional fibre shortening increased from 10 to 33% and the SCI from 26 to 55% in the more severe of the two cases). Hypertrophy and dilatation decreased significantly. This is a so-called intermediary form of carnitine insufficiency and very unusual because of the isolated cardiac involvement. These cases underline the value of systematic muscle biopsy with measurement of carnitine concentrations in the investigation of all cases of supposed primary cardiomyopathy, especially as a rapid improvement can be obtained by specific replacement therapy.

Adolescent↗
PubMed1986

Preleukemia manifested by hemolytic anemia with pyruvate-kinase deficiency.

A 27-year-old patient manifested severe hemolytic anemia during a preleukemia phase. Low levels of adenosine triphosphate and decreased activity of pyruvate kinase were found. Acute myeloblastic leukemia developed in this patient 20 months later. The case suggests that acquired enzyme deficiency may play a role in the development of anemia in the preleukemic state.

Adult↗
PubMed1985

Initiation of transcription by bacteriophage T4-modified RNA polymerase independently of host sigma factor.

After infection of Escherichia coli with bacteriophage T4 a series of modifications of RNA polymerase takes place including the association of several small polypeptides. We isolated RNA polymerase from cells abortively infected with a series of T4 mutants which arrest phage development at different stages and found that different sets of associated proteins are present in RNA polymerase in each case. The patterns of associated polypeptides seem to correlate with DNA content in the infected cells, suggesting that some of them can be involved both in DNA replication and in the transcription apparatus. One of the modified forms of RNA polymerase contains stoichiometric amounts of a protein with Mr = 25,000 (25K protein), which remains associated with the core enzyme after the removal of sigma factor by chromatography on phosphocellulose. The 25K protein was purified to homogeneity and its effect on transcription selectivity was analyzed in an in vitro system using fragments of T4 DNA as templates. The 25K protein exists in two functional forms which direct core RNA polymerase to utilize two different types of transcription start sites (class I and class II promoters). Both activities do not require host sigma factor. The two forms of 25K protein seem to compete with each other for the core enzyme. The isolated 25K protein can form stable dimers, suggesting that its two activities are associated with the dimeric and monomeric forms. Class I (but not class II) promoters can also be utilized in response to the host sigma factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoradiography↗
PubMed1985

[Long-term follow-up of abused children (apropos of 65 cases)].

The authors work in an area located north of Paris, which contains roughly a population of .75 million, of which 82,000 are children less than 3 years of age. The center and main city of the area is the town of Amiens. The recruitment of their cases under study was essentially hospital based, which explains the relative severity of the trauma observed. The children were followed by various observation systems. Seventy-four children, of which more than two-thirds were less than 3 years of age were followed for a period varying from two to ten years. Forty-three percent of these children have been maintained in their biological families; the others have been placed. With this system only nine recurrences of abuse have been observed (12%), and only two instances of death have been observed. Other studies have demonstrated higher proportions. However, in two-thirds of the children observed, there were minor or major psychological or even psychiatric sequelae. Protection services have tried to stay away from institutional placement as much as possible; when necessary, the foster home system or adoption has been used. Striking is the lack of means available to help abusing families. Efforts should be made to assist the abusing families stay together.

Battered Child Syndrome↗
PubMed1985

Hypomethylation of DNA derived from purified human erythroid cells correlates with gene activity of the beta-globin cluster.

Analysis of methylation at the beta-globin gene cluster was carried out on DNA derived from nucleated RBCs (orthochromatic normoblasts) isolated from peripheral blood of patients with beta-thalassemia major or other congenital hemolytic anemia after splenectomy. A procedure to separate these normoblasts from the other nucleated cells of the peripheral blood was developed, providing us with a convenient source of DNA for investigating parameters related to human erythroid differentiation. Blood samples were obtained from six adult patients who express their gamma-globin genes at different levels. Inverse correlation between methylation and gene activity was consistently observed for five of the eight sites analyzed. A site 3' to the beta gene was always unmethylated, two sites flanking the epsilon gene were always found to be methylated, and two sites 5' to the two gamma genes, G gamma and A gamma, were hypomethylated in correlation with gamma gene activity of the individual patients. A site 5' to the delta gene was unmethylated in normoblasts as well as in WBC. No apparent relation between hypomethylation and gene activity was observed for two additional sites. The results suggest that methylation at specific chromosomal locations participate in genetic regulation of the beta-like globin genes in humans.

DNA↗
PubMed1985

Noninvasive analysis of skin iron and zinc levels in beta-thalassemia major and intermedia.

Diagnostic x-ray spectrometry, a method based on x-ray fluorescence analysis, was used for noninvasive determination of iron and zinc in two distinct skin areas, representing predominantly dermal and epidermal tissues, in 56 patients with beta-thalassemia major and intermedia. The mean iron levels in the skin of patients with beta-thalassemia major and intermedia were elevated by greater than 200% and greater than 50%, respectively, compared with control values. The zinc levels of both skin areas examined were within the normal range. The data indicate that the rate and number of blood transfusions, which correlated well with serum ferritin levels (r = 0.8), are not the only factors that determine the amount of iron deposition in the skin (r less than 0.6). Other sources of iron intake contribute to the total iron load in the tissues, particularly in patients who are not given multiple transfusions. The noninvasive quantitation of skin levels may reflect the extent of iron deposition in major parenchymal organs. Repeated DXS examinations of the skin could monitor the clearance of iron from the tissues of patients with iron overload in the course of therapy with chelating agents.

Blood Transfusion↗
PubMed1984

The effect of a bacteriophage T4-induced polypeptide on host RNA polymerase interaction with promoters.

After infection of Escherichia coli with bacteriophage T4, the host RNA polymerase acquires several small phage-induced polypeptides (Stevens, A. (1974) Biochemistry 13, 493-503) and its alpha subunits get ADP-ribosylated by a virus-specific enzyme (Zillig, W., Mailhammer, R., Skorko, R., and Rohrer, H. (1977) Curr. Top. Cell. Regul. 12, 263-271). The modified polymerase displays changed enzymatic properties including sensitivity to increased salt concentration and a higher transition temperature of open promoter complex formation (promoter melting temperature). In order to assess the role of individual modifications in the changed enzyme properties, we isolated RNA polymerase from cells infected with T4 mutant defective in the ADP-ribosylating enzyme. We also purified one of the associated polypeptides, the 15,000-dalton protein which is invariably present in stoichiometric amounts in different RNA polymerase preparations. In an in vitro transcription system using T4 DNA as template, we demonstrate that the 15-kDa protein is the cause of the elevated promoter melting temperature and can induce this property when added to host RNA polymerase. We also show that the increased salt sensitivity of T4-modified polymerase is primarily the result of ADP-ribosylation of its alpha subunits.

Adenosine Diphosphate Ribose↗