Biomedical subjects

A Goldfarb

Publications and source records attributed to A Goldfarb.

At least 73 records · Page 4Linked to original sources

PubMed1994

Diversity of beta-globin mutations in Israeli ethnic groups reflects recent historic events.

We characterized nearly 500 beta-thalassemia genes from the Israeli population representing a variety of ethnic subgroups. We found 28 different mutations in the beta-globin gene, including three mutations (beta S, beta C, and beta O-Arab) causing hemoglobinopathies. Marked genetic heterogeneity was observed in both the Arab (20 mutations) and Jewish (17 mutations) populations. On the other hand, two ethnic isolates--Druze and Samaritans--had a single mutation each. Fifteen of the beta-thalassemia alleles are Mediterranean in type, 5 originated in Kurdistan, 2 are of Indian origin, and 2 sporadic alleles came from Europe. Only one mutant allele--nonsense codon 37--appears to be indigenous to Israel. While human habitation in Israel dates back to early prehistory, the present-day spectrum of beta-globin mutations can be largely explained by migration events that occurred in the past millennium.

Alleles↗

PubMed1993

Two modes of transcription initiation in vitro at the rrnB P1 promoter of Escherichia coli.

The rrnB P1 promoter of Escherichia coli (starting sequence C-4-A-3-C-2-C-1-A+1-C+2-U+3-G+4) forms a binary complex with RNA polymerase that is highly unstable and requires the presence of transcription substrates ATP and CTP for stabilizing the enzyme-DNA association (Gourse, R. L. (1988) Nucleic Acids Res. 16, 9789-9809). We show that in the absence of UTP and GTP the stabilization is accomplished by short RNA oligomers synthesized in an unusual "-3-->" mode whereby the primer initiated at the +1 site presumably slips back by three nucleotides into the -3 site and is then extended yielding stable ternary complexes. By contrast, short oligomers initiated in the conventional "+1-->" mode without slippage do not exert the stabilization effect and are readily aborted from the promoter complex. The stable -3-->ternary complexes carry sigma factor but otherwise resemble elongation complexes in their high salt stability and in the fact that they are formed with a mutant RNA polymerase deficient in promoter binding. A model is proposed explaining the stability of the -3-->ternary complexes by RNA slipping into a putative "tight RNA binding site" in RNA polymerase which is normally occupied by RNA during elongation.

Cold Temperature↗

PubMed1993

Bacteriophage T4 Alc protein: a transcription termination factor sensing local modification of DNA.

Bacteriophage T4 Alc protein participates in shutting off host transcription after infection of E. coli. It is demonstrated that Alc acts as a site-specific termination factor. The Alc sites occur frequently in E. coli DNA, resulting in early cessation of elongation in several tested transcription units. Alc-dependent termination requires unimpeded propagation of the elongating complex as it approaches the Alc site. Temporary halting of RNA polymerase within 10-15 bp before the Alc site prevents termination. Bacteriophage T4 transcription is protected from the action of Alc by overall substitution of cytosine with 5-hydroxymethyl cytosine in T4 DNA. In vitro methylation of CpG sequences in the vicinity of an Alc site abolishes the effect of Alc. Thus, Alc-dependent termination involves local sensing of the state of cytosine modification and a short-term "memory" of recent pausing.

Bacteriophage T4↗

PubMed1993

Active center rearrangement in RNA polymerase initiation complex.

His1237 in the beta subunit of Escherichia coli RNA polymerase marks the "5' face" of the active center since it can be cross-linked to the gamma-phosphate of the priming substrate. It is demonstrated that RNA chains up to 9 nucleotides in length can be synthesized using His1237-cross-linked nucleotide as a primer. Thus, a substantial mass of RNA can be accommodated in the active center between His1237 and the site of catalysis that remains juxtaposed to the growing 3' end. The apparent "filling" of the active center with RNA precedes promoter clearance and suggests a mechanism of coupling between catalysis and saltatory translocation of RNA polymerase.

Amino Acid Sequence↗

PubMed1993

Histidine-tagged RNA polymerase: dissection of the transcription cycle using immobilized enzyme.

A stretch of six histidine residues (His6) has been genetically fused to the C terminus of the beta' polypeptide of Escherichia coli RNA polymerase. The His6-tagged beta' subunit assembles into RNA polymerase molecules which perform all vital in vivo functions and behave qualitatively normally in vitro. The His6 tag permits rapid purification of the enzyme directly from crude cell extracts or from an in vitro reconstitution reaction by adsorption to Ni(2+)-chelating agarose resin, followed by elution with imidazole. The enzyme bound to the matrix remains transcriptionally active. The immobilized enzyme can withstand repeated buffer changes without substantial activity loss and permits controlled stepwise 'walking' of the transcriptional complex along the DNA template, and isolation of defined intermediates in the transcription cycle. The immobilized RNA polymerase provides a powerful experimental system for structural and functional analysis of RNA polymerase and its interaction with regulatory factors.

Amino Acid Sequence↗

PubMed1993

Rifampicin region revisited. New rifampicin-resistant and streptolydigin-resistant mutants in the beta subunit of Escherichia coli RNA polymerase.

Mutations to rifampicin resistance (RifR) in Escherichia coli alter the beta subunit of RNA polymerase (RNAP). A series of new point RifR mutations was isolated with the aid of random polymerase chain reaction-mediated mutagenesis followed by selection on rifampicin (Rif). All of the new RifR mutants, including changes in two novel positions, fell into the two principal clusters previously identified in the middle section of beta. Disruption of the spacer between the two clusters with deletions and insertions led to RNAP that was functional and sensitive to Rif in vitro, indicating that the spacer is not directly involved in Rif binding. However, most of the spacer mutants were strongly resistant to streptolydigin, suggesting that this area is involved in the binding of streptolydigin. An insertion of six consecutive histidines into the spacer was constructed that could be used to anchor RNAP on a Nichelate matrix without the loss of enzymatic activity, indicating that this region is looped out of the RNAP molecule.

Amino Acid Sequence↗

PubMed1993

Transcript cleavage factors from E. coli.

Two transcription elongation factors (GreA and GreB) related in primary sequence were isolated from E. coli. Each factor induced cleavage of the nascent transcript in artificially halted elongation complexes followed by the loss of the 3' proximal fragment and resumption of elongation from the new 3' terminus. GreA induced cleavages 2 or 3 nt behind the terminus while GreB released longer oligonucleotides up to 9 nt in length. The pattern of cleavages characteristically changed as the transcription complex advanced, supporting the "inchworm" model of RNA polymerase propagation. In addition to attacking artificially halted complexes, both factors antagonized the action of natural elongation-arresting sites that occasionally trap the advancing complex. GreB rescued the arrested complexes via the transcript cleavage and restart pathway while GreA acted by an unknown mechanism, preventing the arrest only if added before the polymerase reached the arresting site.

Amino Acid Sequence↗

PubMed1993

Dominant lethal mutations near the 5' substrate binding site affect RNA polymerase propagation.

The segment Asp1064-Lys1073 in the beta subunit of Escherichia coli RNA polymerase is evolutionarily conserved and is located near the "5' face" of the nucleotide binding pocket as was shown by affinity labeling with priming substrates (Grachev, M. A., Lukhtamov, E. A., Mustaev, A. A., Zaychikov, E. F., Abdukayumov, M. N., Rabinov, I. V., Richter, V. I., Skoblov, Y. S., and Chistyakov, P. G. (1989) Eur. J. Biochem. 180, 577-585). We engineered single Xaa-->Ala or Ala-->Ser substitutions of eight evolutionarily conserved amino acids in this segment as well as a multiple alanine (KRNK) substitution of four of these residues. The KRNK mutation as well as four of the single substitutions were dominant lethal, two of the single mutations were recessive lethal, and two were viable. RNA polymerase bearing the dominant mutations was prepared for biochemical study by in vitro reconstitution from subunits. All of the mutant enzymes formed stable, specific promoter complexes, capable of initiating RNA synthesis. However, the KRNK polymerase was totally blocked in initiation-to-elongation transition, whereas the four point mutants displayed allele-specific changes in promoter clearance rate. Each of the four mutations changed, in a specific way, both the pattern of short oligomers generated in abortive initiation and the pattern of RNA polymerase pausing during elongation. Thus, the mutations appear to distort but not destroy the active center and to alter, in allele-specific manner, the coupling between the catalytic reaction and RNA polymerase propagation along the template.

Alleles↗

PubMed1993

Recombinant Escherichia coli RNA polymerase: purification of individually overexpressed subunits and in vitro assembly.

New improved methods were developed for the purification to apparent homogeneity of alpha, beta, beta', and sigma subunits of Escherichia coli RNA polymerase (RNAP) from corresponding overproducing strains. The purified subunits were assembled into enzymatically active RNAP holoenzyme (alpha 2 beta beta' sigma) using the optimal subunit molar ratio (alpha:beta:beta':sigma = 2:8:4:1) at a total protein concentration of 0.5 mg/ml. The presence of sigma subunit and 10 microM ZnCl2 in the reconstitution mixture increased the yield of RNAP approximately 4 times. The assembled RNA polymerase was purified by two successive chromatographic steps using size-exclusion Superose 6 and anion exchange Mono Q FPLC columns, which resulted in the electrophoretically homogeneous holoenzyme with overall yield of 56%. The specific activity of the recombinant RNAP estimated by the standard T4 transcription assay was 6.5 nmol of [3H]UTP incorporated into acid-insoluble RNA product per microgram of RNAP per 1 h.

Bacterial Proteins↗

PubMed1993

Impaired neutrophil chemotaxis in patients with thalassaemia major.

Random and directed migration, O2- production, degranulation and adhesion were studied in neutrophils obtained from patients with homozygous beta-thalassaemia and iron overload, in the presence or absence of thalassaemic serum. The only significant defect found was an impairment in directed chemotaxis, further depressed after addition of thalassaemic serum. The chemotactic defect was encountered in all the patients that have suffered from pyogenic infections except one, and was not correlated with the severity of the iron overload. It is suggested that the described neutrophil migration impairment may contribute to the tendency towards infection in certain patients with homozygous beta-thalassaemia.

Adolescent↗

PubMed1993

Cross-sectional and longitudinal study of the pituitary-thyroid axis in patients with thalassaemia major.

OBJECTIVE AND DESIGN: Thyroid dysfunction is known to occur frequently in thalassaemia major, but its prevalence and severity varies in different cohorts, and the long-term natural history is poorly described. We evaluated the pituitary/thyroid axis in thalassaemia major patients in a cross-sectional study and correlated abnormalities with indices of iron overload. Furthermore, the course of thyroid disease in thalassaemia major patients was assessed in a 15-year longitudinal study. PATIENTS AND MEASUREMENTS: Cross-sectional study: pituitary-thyroid axis function was examined in 37 patients (22 F, 15 M; aged 10-39 years, mean +/- SE = 21 +/- 1.4) out of a total of 43 who attended the Haematology and Endocrinology Clinics of Hadassah Hospital on a regular basis. The mean pretransfusion Hb level was 85 +/- 20 g/l, and all patients except one were treated with desferrioxamine (DF, mean +/- SE dose 20.2 +/- 2.6 mg/kg/day). Twenty-two had hypogonadotrophic hypogonadism (HH). Longitudinal study: 21 thalassaemia major patients were evaluated with TRH tests in 1976 and again in 1985. Fourteen of these and another eight were evaluated in both 1985 and 1991. RESULTS: Cross-sectional study: no patient had any clinical signs or symptoms of hypothyroidism; however, one had abnormally low T4, borderline low FT4 and normal T3 levels associated with an exaggerated TSH response to TRH consistent with mild hypothyroidism. This patient did not have a previous TRH test, but serial basal determinations over 7 years revealed a progressive decrease in thyroid function. Thirty-six patients had thyroid hormone levels within the normal range. Nine of these (24.3%) had only an exaggerated TSH response to TRH whereas seven others (19%) also had borderline elevated basal TSH levels. TSH response to TRH was not correlated with age, serum ferritin or liver function tests (ALT or GGT). Longitudinal study: mean TSH response to TRH decreased (P < 0.002), and mean T3 levels increased (P < 0.001) between 1975 and 1985. These findings are probably related to the initiation of DF treatment in 1981. During the last 6 years, four patients with previously normal TSH responses to TRH developed elevated peak TSH levels. Mean T3 concentrations decreased and TSH response to TRH increased significantly (P < 0.001 for both). CONCLUSIONS: (1) In this patient group the thyroid pituitary axis is less sensitive than the gonadal axis to iron-induced damage; only one out of 37 patients developed mild uncompensated hypothyroidism. (2) As opposed to the gonadal axis, the thyroid gland appears to fail before the central components of the axis. (3) Abnormal thyroid function may be reversible in the early stages. (4) Progression is variable, and it may take years to progress from normal to uncompensated hypothyroidism.

Adolescent↗

PubMed1993

A chronic hypercoagulable state and life-long platelet activation in beta thalassemia major.

Increased frequency of thromboembolic events has been recently observed in patients with thalassemia major (TM), causing hypoxemia and cor pulmonale. Autopsy findings demonstrated "old" and recent pulmonary and renal infarcts as well as premature atherosclerosis. Studies to determine hypercoagulability showed: impaired platelet aggregation, increased circulating platelet aggregates, shortened platelet survival, enhanced excretion of urinary metabolites of thromboxane A2 (TXA2) and prostacyclin and decreased plasma levels of Protein C, Protein S or anti-thrombin III. Erythrocytes from TM patients enhanced thrombin formation in a "prothrombinase" assay (using a chromogenic substrate). Chronic anti-thrombotic therapy may be indicated in thalassemic patients to prevent the cardiac and pulmonary complications.

Blood Platelet Disorders↗

PubMed1993

Introduction: oxidant stress, aging, and exercise.

In recent years research in the basic and applied sciences has broadened our understanding of oxygen chemistry and its influences on biological systems. Although living organisms are endowed with a broad array of biochemical defense mechanisms for protection against potentially harmful radical chemistry, oxidative stress may overwhelm those defenses. It is now clear that exercise may initiate oxidative stress. It is the aim of this symposium to review and broaden our understanding of the problem of free radical chemistry as it applies to the exercise milieu.

Aging↗

PubMed1992

Genetic dissection of the transcription cycle. A mutant RNA polymerase that cannot hold onto a promoter.

Deletion of 10 amino acids from a conserved motif in the beta subunit of Escherichia coli RNA polymerase (RNAP) leads to an interrupted transcription cycle and lethal phenotype. RNAP carrying the mutant subunit retains catalytic function and specificity of promoter recognition but is unable to efficiently hold onto DNA in the binary complex, resulting in a diminished initiation frequency. However, inefficient initiation by the mutant enzyme leads to processive and stable ternary elongating complex. Thus, the mutation dissects the traits of promoter selectivity, binary complex stability, and ternary complex processivity reflecting compartmentalization of function within the RNAP molecule.

Amino Acid Sequence↗

PubMed1992

GreA protein: a transcription elongation factor from Escherichia coli.

A protein identified as the 158-amino acid product of the greA gene was isolated from Escherichia coli. When added to a halted ternary transcription complex, the GreA protein induced cleavage and removal of the 3' proximal dinucleotide from the nascent RNA. The new 3' terminus generated by the cleavage could be extended into longer transcripts. GreA-mediated cleavage of a transcript appears to permit a ternary complex to resume transcription from a state of indefinite elongation arrest induced by a specific DNA site. The GreA protein tended to interact with RNA polymerase during purification and recycled between RNA polymerase molecules in the course of the in vitro cleavage reaction. Similar biochemical activities have been reported in eukaryotic RNA polymerases, indicating that transcript cleavage and restart of elongation may be a general transcriptional mechanism.

Bacterial Proteins↗

PubMed1992

Primaquine-induced superoxide production by beta-thalassemic red blood cells.

Primaquine, a prooxidant antimalarial drug, incubated with human red blood cells (RBC) induced marked superoxide generation in the cells as detected by exogenous cytochrome c reduction. In the presence of primaquine, beta-thalassemic RBC produced significantly more superoxide than normal RBC, thus reflecting the vulnerability of beta-thalassemic cells to oxidative stress.

Erythrocytes↗

PubMed1992

Dissection of the beta subunit in the Escherichia coli RNA polymerase into domains by proteolytic cleavage.

The 1342 amino acid long beta subunit of Escherichia coli RNA polymerase includes a dispensable region (residues 940-1040) that is absent in homologous RNA polymerase subunits from chloroplasts, eukaryotes, and archaebacteria (Borukhov, S., Severinov, K., Kashlev, M., Lebedev, A., Bass, I., Rowland, G. C., Lim, P.-P., Glass, R. E., Nikiforov, V., and Goldfarb, A. (1991) J. Biol. Chem. 266, 23921-23926). Genetic disruption of this region by in-frame deletion or insertion sensitizes the beta subunit in assembled RNA polymerase molecules to attack by trypsin. We demonstrate that RNA polymerase with the beta polypeptide cleaved in the dispensable region retains normal in vitro activity. Moreover, the RNA polymerase activity is completely restored after denaturation and reconstitution of the enzyme carrying cleaved beta subunit indicating that its carboxyl- and amino-terminal parts fold and assemble into RNA polymerase as separate entities.

Amino Acid Sequence↗

PubMed1992

Effects of coenzyme athletic performance system as an ergogenic aid on endurance performance to exhaustion.

This study examined the effects of the Coenzyme Athletic Performance System (CAPS) on endurance performance to exhaustion. CAPS contains 100 mg coenzyme Q10, 500 mg cytochrome C, 100 mg inosine, and 200 IU vitamin E. Eleven highly trained male triathletes were given three daily doses of either CAPS or placebo (dicalcium phosphate) for two 4-week periods using a double-blind crossover design. A 4-week washout period separated the two treatment periods. An exhaustive performance test, consisting of 90 minutes of running on a treadmill (70% VO2max) followed by cycling (70% VO2max) until exhaustion, was conducted after each treatment period. The mean (+/- SEM) time to exhaustion for the subjects using CAPS (223 +/- 17 min) was not significantly different (p = 0.57) from the placebo trial (215 +/- 9 min). Blood glucose, lactate, and free fatty acid concentrations at exhaustion did not differ between treatments (p < 0.05). CAPS had no apparent benefit on exercise to exhaustion.

Adolescent↗