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Biomedical subjects

A Goldfarb

Publications and source records attributed to A Goldfarb.

At least 19 recordsLinked to original sources
PubMed1999

Disruption of substrate binding site in E. coli RNA polymerase by lethal alanine substitutions in carboxy terminal domain of the beta subunit.

Alanine substitution of four amino acids in two evolutionarily conserved motifs, PSRM and RFGEMIE, near the carboxy terminus of the beta subunit of E. coli RNA polymerase results in a dramatic loss of the enzyme's affinity to substrates with no apparent effect on the maximal rate of the enzymatic reaction or on binding to promoters. The magnitude and selectivity of the effect suggest that the mutations disrupt the substrate binding site of the active center.

Binding Sites↗
PubMed1998

Spatial organization of transcription elongation complex in Escherichia coli.

During RNA synthesis in the ternary elongation complex, RNA polymerase enzyme holds nucleic acids in three contiguous sites: the double-stranded DNA-binding site (DBS) ahead of the transcription bubble, the RNA-DNA heteroduplex-binding site (HBS), and the RNA-binding site (RBS) upstream of HBS. Photochemical cross-linking allowed mapping of the DNA and RNA contacts to specific positions on the amino acid sequence. Unexpectedly, the same protein regions were found to participate in both DBS and RBS. Thus, DNA entry and RNA exit occur close together in the RNA polymerase molecule, suggesting that the three sites constitute a single unit. The results explain how RNA in the integrated unit RBS-HBS-DBS may stabilize the ternary complex, whereas a hairpin in RNA result in its dissociation.

Binding Sites↗
PubMed1998

Cost-benefit analysis of a national thalassaemia prevention programme in Israel.

OBJECTIVE: In Israel (population 5.7 million) there are around 200 known living subjects with thalassaemia major, of whom around 80% are from the northern district. This study aims at examining the costs and benefits of a national screening programme to prevent thalassaemia in Israel. MEASUREMENTS AND MAIN RESULTS: The lifetime healthcare costs of caring for a person born with thalassaemia major are $284,154. The costs of the home infusion service (33.1%) actually exceed the costs of the chelating agent itself (22.1%). The remaining 44.8% of costs are due to stay in hospital, operations, outpatient visits, laboratory tests, therapists, etc. Lost earnings and premature mortality costs account for a further $51,843 and $141,944 respectively for each case. A national screening programme would cost $900,197 and prevent around 13.4 homozygotes being born, at a cost of $67,369 for each birth prevented. The benefit-cost ratio of the programme to the health services is 4.22:1, which increases to 6.01:1 when a societal perspective is taken. However, around 13.0 homozygote births are still expected to occur, the majority owing to lack of compliance of patients at various stages in the screening process. The addition of a national health education programme for the higher risk non-Jewish population either nationally or in selected regions will incur extra costs, which may be covered by increased benefits as a result of better compliance with the screening programme. CONCLUSION: Israel should start to provide a nationwide thalassaemia screening programme as the monetary benefits to society (and even to the health services alone) will exceed the screening programmes costs.

Cost-Benefit Analysis↗
PubMed1998

Heme arginate therapy for beta thalassemia: in vitro versus in vivo effects.

Hemin has a profound effect on erythroid cell maturation and promotes fetal hemoglobin synthesis in vitro. In beta-thalassemia, increasing fetal hemoglobin levels can ameliorate the anemia. We administered heme arginate, a novel stable form of hemin, to 4 patients with thalassemia intermedia and studied the in vitro versus in vivo effects. In erythroid cultures, there was a marked rise in total hemoglobin and hemoglobin F. In vivo, 3 of 4 patients had a rise in hemoglobin levels (from 0.4 to 1.1 g%), which was statistically significant in 1 patient. There were no serious adverse effects. Heme arginate may be useful in the treatment of thalassemia intermedia.

Adult↗
PubMed1998

Recombinant von Willebrand factor fragment AR545C inhibits platelet aggregation and enhances thrombolysis with rtPA in a rabbit thrombosis model.

Platelet adhesion to exposed subendothelium is mediated by platelet receptor glycoprotein Ib and polymeric von Willebrand factor (vWF). To improve the results of coronary arterial thrombolysis, fragments of vWF with enhanced glycoprotein Ib binding competitive with native vWF have been proposed as adjuvants to recombinant tissue-type plasminogen activator (rtPA). We designed a recombinant vWF fragment spanning Ala444 to Asp730 that contains the Arg545Cys mutation (named AR545C) and analyzed its antiplatelet properties in vitro and in vivo. AR545C-platelet interaction was assessed by ristocetin or botrocetin-induced platelet agglutination, or interaction with extracellular matrix under arterial flow conditions. AR545C showed enhanced reactivity with platelet glycoprotein Ib at low concentrations of ristocetin, and 60% bound spontaneously to platelets. AR545C inhibited ristocetin-induced platelet agglutination in a dose-dependent manner, with a concentration necessary to inhibit 50% of agglutination of 0.16+/-0.04 micromol/L. The inhibitory effect of AR545C on rabbit botrocetin-induced platelet agglutination was also dose dependent, with a concentration necessary to inhibit 50% of agglutination of 0.3 to 0.5 micromol/L. AR545C also completely inhibited aggregate formation and decreased the adhesion of platelets to extracellular matrix by 62.5%. The effect of AR545C on thrombolysis with rtPA was evaluated using a modified rabbit femoral thrombosis model. Local injection of AR545C into the thrombosed segment of rabbit femoral artery significantly shortened the time to reperfusion with rtPA (60+/-17.3 versus 103+/-15.2 minutes, P=.05) and significantly prolonged the total patency time (175 versus 21 minutes, P=.04). No significant difference was found in the reperfusion rate or time to reocclusion. AR545C is a potential antithrombotic agent that enhances the thrombolytic effect of rtPA in the rabbit model.

Animals↗
PubMed1997

Modular organization of the catalytic center of RNA polymerase.

The Fe2+ ion that specifically replaces Mg2+ in the active center of RNA polymerase generates reactive hydroxyl radicals that cause highly localized cleavage of polypeptide chains. Mapping of the cleavage sites revealed the overall architecture of the active center. Nine distinct sites, five in the beta subunit and four in the beta' subunit of Escherichia coli RNA polymerase, all at or near highly conserved sequence motifs, are brought together in the enzyme's ternary structure within the distance of approximately 1 nm from the active center Me2+. These sites are located in at least six different domains of the subunits, reflecting modular organization of the active center.

Amino Acid Sequence↗
PubMed1997

The RNA-DNA hybrid maintains the register of transcription by preventing backtracking of RNA polymerase.

An 8-9 bp RNA-DNA hybrid in the transcription elongation complex is essential for keeping the RNA 3' terminus engaged with the active site of E. coli RNA polymerase (RNAP). Destabilization of the hybrid leads to detachment of the transcript terminus, RNAP backtracking, and shifting of the hybrid upstream. Eventually, the exposed 3' segment of RNA can be removed through transcript cleavage. At certain sites, cycles of unwinding-rewinding of the hybrid are coupled to reverse-forward sliding of the transcription elongation complex. This explains apparent discontinuous elongation, which was previously interpreted as contraction and expansion of an RNAP molecule (inch-worming). Thus, the 3'-proximal RNA-DNA hybrid plays the dual role of keeping the active site in register with the template and sensing the helix-destabilizing mismatches in RNA, launching correction through backtracking and cleavage.

Base Sequence↗
PubMed1997

Genetic analysis of beta-thalassemia intermedia in Israel: diversity of mechanisms and unpredictability of phenotype.

Molecular analysis was performed on 95 Israeli patients with thalassemia intermedia, representing 60 families of Arab (Moslem and Christian), Jewish, Druze, and Samaritan origin. There was a wide range of phenotypic severity, with baseline hemoglobin levels ranging from 5.5 to 10.7. Eighteen thalassemia mutations were found (29 genotypes), which were subdivided into groups, according to the severity of mutations. A consistently mild phenotype (10 families) was caused by compound heterozygosity for a silent mutation, such as -101 C-T or by coexistence of triplicated alpha-globin genes with thalassemia trait. In 39 thalassemia intermedia families, the genotype which was found was one which led to severe thalassemia intermedia, or, in other families, was associated with thalassemia major. Elevated hemoglobin F ameliorated the disease in some patients with a severe genotype. We did not find a beneficial effect of concurrent alpha-thalassemia in any of the families studied. In 11 families, only one beta-thalassemia allele was identified. One was a dominant thalassemia intermedia allele. Three additional families with heterozygous beta-thalassemia had excess alpha-globin genes (5 or 6 total). In 7 of these heterozygotes, no explanation was found for the thalassemia intermedia phenotype. Our results suggest a substantial influence of as yet unknown genetic modifiers. These findings have important implications for prenatal diagnosis and for the genetic counseling of families with thalassemia intermedia.

Alleles↗
PubMed1997

In-vivo platelet activation correlates with red cell anionic phospholipid exposure in patients with beta-thalassaemia major.

Several clinical and laboratory findings suggest the presence of a chronic hypercoagulable state in patients with beta-thalassaemia major (TM). We have previously shown that isolated TM red blood cells (RBC) strongly enhance prothrombin activation, suggesting an increased membrane exposure of procoagulant phospholipids (i.e. phosphatidylserine). In this study we quantitated the procoagulant activity of RBC in TM and thalassaemia intermedia (TI) patients. We also determined the fraction of activated platelets expressing p-selectin (CD62p) or CD63 in these subjects. Both assays were performed by dual-colour flow cytometry. A significantly (P < 0.01) higher fraction of FITC-annexin V-labelled RBC was found in TM and TI patients, compared to the controls. A highly significant correlation (P < 0.001) was found in TM patients between the number of RBC-bound annexin V molecules and the fraction of CD62p (p-selectin) or CD63-positive platelets. This association between annexin V binding to TM RBC and the expression of platelet activation markers was also found in individual TM patients over time. Thus, the procoagulant surface of TM RBC may accelerate thrombin generation in vivo which, in turn, triggers platelet activation.

Adolescent↗
PubMed1996

Structural modules of the large subunits of RNA polymerase. Introducing archaebacterial and chloroplast split sites in the beta and beta' subunits of Escherichia coli RNA polymerase.

The beta and beta' subunits of Escherichia coli DNA-dependent RNA polymerase are highly conserved throughout eubacterial and eukaryotic kingdoms. However, in some archaebacteria and chloroplasts, the corresponding sequences are "split" into smaller polypeptides that are encoded by separate genes. To test if such split sites can be accommodated into E. coli RNA polymerase, subunit fragments encoded by the segments of E. coli rpoB and rpoC genes corresponding to archaebacterial and chloroplast split subunits were individually overexpressed. The purified fragments, when mixed in vitro with complementing intact RNA polymerase subunits, yielded an active enzyme capable of catalyzing the phosphodiester bond formation. Thus, the large subunits of eubacteria and eukaryotes are composed of independent structural modules corresponding to the smaller subunits of archaebacteria and chloroplasts.

Amino Acid Sequence↗
PubMed1996

Transcription processivity: protein-DNA interactions holding together the elongation complex.

The elongation of RNA chains during transcription occurs in a ternary complex containing RNA polymerase (RNAP), DNA template, and nascent RNA. It is shown here that elongating RNAP from Escherichia coli can switch DNA templates by means of end-to-end transposition without loss of the transcript. After the switch, transcription continues on the new template. With the use of defined short DNA fragments as switching templates, RNAP-DNA interactions were dissected into two spatially distinct components, each contributing to the stability of the elongating complex. The front (F) interaction occurs ahead of the growing end of RNA. This interaction is non-ionic and requires 7 to 9 base pairs of intact DNA duplex. The rear (R) interaction is ionic and requires approximately six nucleotides of the template DNA strand behind the active site and one nucleotide ahead of it. The nontemplate strand is not involved. With the use of protein-DNA crosslinking, the F interaction was mapped to the conserved zinc finger motif in the NH2-terminus of the beta' subunit and the R interaction, to the COOH-terminal catalytic domain of the beta subunit. Mutational disruption of the zinc finger selectively destroyed the F interaction and produced a salt-sensitive ternary complex with diminished processivity. A model of the ternary complex is proposed here that suggests that trilateral contacts in the active center maintain the nonprocessive complex, whereas a front-end domain including the zinc finger ensures processivity.

Base Sequence↗
PubMed1996

Mapping of catalytic residues in the RNA polymerase active center.

When the Mg2+ ion in the catalytic center of Escherichia coli RNA polymerase (RNAP) is replaced with Fe2+, hydroxyl radicals are generated. In the promoter complex, such radicals cleave template DNA near the transcription start site, whereas the beta' subunit is cleaved at a conserved motif NADFDGD (Asn-Ala-Asp-Phe-Asp-Gly-Asp). Substitution of the three aspartate residues with alanine creates a dominant lethal mutation. The mutant RNAP is catalytically inactive but can bind promoters and form an open complex. The mutant fails to support Fe2+-induced cleavage of DNA or protein. Thus, the NAD-FDGD motif is involved in chelation of the active center Mg2+.

Amino Acid Sequence↗
PubMed1996

Protein-RNA interactions in the active center of transcription elongation complex.

By using a crosslinkable probe incorporated into the 3' terminus of nascent transcript, three sites were mapped in Escherichia coli RNA polymerase that are contacted by the RNA in the productive elongation complex. Two of these sites are in the beta subunit and one is in the beta' subunit. During elongation, the transcription complex occasionally undergoes an arrest whereby it can neither extend nor release the RNA transcript. It is demonstrated that in an arrested complex, the three contacts of RNA 3' terminus are lost, while a new beta' subunit contact becomes prominent. Thus, elongation arrest appears to involve the disengagement of the bulk of the active center from the 3' terminus of RNA and the transfer of the terminus into a new protein environment.

Affinity Labels↗
PubMed1996

Surgical treatment of vulvar lichen sclerosus: a review.

Surgical therapy of lichen sclerosus of the vulva consists of three main operations: vulvectomy (with or without a skin graft), cryosurgery, and laser ablation. It is indicated in two conditions, either when malignant transformation is present or is likely to occur, or when medical treatment has failed. The overall risk for malignant transformation of vulvar lichen sclerosus is low, ranging between 0 and 9 percent. However, specific histological criteria, such as mixed dystrophy, have recently been associated with higher malignancy rates. Vulvectomy is indicated only when these criteria are met. Both skinning and simple vulvectomies are associated with recurrence rates as high as 50 percent. However, better sexual function and cosmetic results have been reported in the former, especially with concomitant split skin grafting. Cryosurgery also has high recurrence rates, although short-term results are favorable. Although only small series have been reported, laser therapy seems to carry better long-term results than other modes of treatment. Convalescence is complete within 6 weeks posttreatment, and remission rates are as high as 85 percent at 3 years of follow-up. The high recurrence rate of all surgical modalities makes surgical treatment suitable only for patients who failed to respond to multiple medical treatments such as topical high potent steroid ointments, testosterone, and retinoids.

Adult↗
PubMed1996

Enhanced aggregability of red blood cells of beta-thalassemia major patients.

beta-Thalassemia major (TM), a congenital hemoglobinopathy, is associated with hemodynamic disorders and with structural red blood cell (RBC) anomalies that may indicate impairment of RBC rheological properties. To gain insight into the possible contribution of RBC to the hemodynamic disorders, we studied RBC aggregability, which plays a central role in blood flow, particularly in the microcirculation. RBC aggregate size distribution and morphology of TM RBC were determined using a novel system for image analysis of blood cells in a flow chamber. It was found that the aggregability of RBC of TM patients is markedly enhanced. These cells form large clusters, as opposed to normal rouleaux, and higher shear stress is required to disperse them. The aggregate size of TM RBC is reduced to the normal range after the patients have received a blood transfusion. This study suggests that the hemodynamic disorders observed in TM may be linked to the enhanced RBC aggregability and that improvement of RBC rheological properties may be considered in the treatment of thalassemia.

Adolescent↗