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A Goldman

Publications and source records attributed to A Goldman.

At least 37 records · Page 2Linked to original sources
PubMed2001

Immunoaffinity purification and reconstitution of human alpha(2)-adrenergic receptor subtype C2 into phospholipid vesicles.

Large quantities of correctly folded, pure alpha(2)-adrenergic receptor protein are needed for structural analysis. We report here the first efficient method to purify human alpha(2)-adrenergic receptor subtype C2 to homogeneity from recombinant yeast Saccharomyces cerevisiae by one-step purification using a monoclonal antibody column (specific for alpha(2)C2). We show that the adrenoceptor antagonist phentolamine stabilized the receptor during purification. We used a very effective chaotropic agent, NaSCN, to elute the receptor from the immunoaffinity column with an overall yield of 34% before reconstitution. Ligand binding of detergent-solubilized, immunoaffinity-purified receptors could not be demonstrated, but partial recovery of ligand binding activity was achieved when purified receptors were reconstituted into phospholipid vesicles. The reconstituted receptors still bound radioligand after storage on ice for 4 weeks. This purification procedure can be easily scaled-up and thus demonstrates the utility of a monoclonal antibody column and NaSCN elution to purify large quantities of G-protein-coupled receptors.

Adrenergic alpha-2 Receptor Antagonists↗
PubMed2001

Comparison of two calcium blockers on hemodynamics, left ventricular mass, and coronary vasodilatory in advanced hypertension.

Dihydropyridine and nondihydropyridine calcium channel blockers (CCB) differ in pharmacologic characteristics. Few clinical studies distinguish effects of CCB as monotherapy. We conducted a comprehensive comparison of two CCB on patients with moderate to severe hypertension. Thirty patients with pretreatment diastolic blood pressures > or = 100 mm Hg were randomly assigned to either nifedipine-GITS or verapamil-SR. Dose titration achieved a diastolic blood pressure of < or = 95 mm Hg or a decrease of > or = 15 mm Hg over 4 weeks. Clinic blood pressure (BP), 24-h ambulatory BP, exercise BP, left ventricular mass, systolic and diastolic function by echocardiography, and coronary flow reserve by split-dose thallium-201 imaging with adenosine were assessed at baseline, end of titration, 3 months and 6 months of treatment. Plasma renin activity, atrial natriuretic peptide, norepinephrine, and epinephrine were assayed. Both drugs caused similar reductions in clinic and 24-h ambulatory BP and similar reductions in left ventricular mass index. Compared to nifedipine-GITS, verapamil-SR produced a significantly lower resting and peak exercise heart rate. Nifedipine-GITS elicited a lower peak exercise systolic BP. At end titration nifedipine-GITS produced lower plasma atrial natriuretic peptide levels, no longer apparent by 6 months. Plasma norepinephrine was lower with verapamil-SR, also at end titration and at 3 months, but not at 6 months. Plasma epinephrine and plasma renin activity were unchanged by either drug. There was no difference for systolic or diastolic left ventricular function or coronary flow reserve between the two treatments. Once daily nifedipine-GITS and verapamil-SR are equally effective for reduction of arterial pressure in moderate to severe hypertension. Differences in their hemodynamic profiles and neurohormonal responses are consistent with preclinical pharmacologic characteristics. The clinical implications of their similarities and differences remain to be fully evaluated in outcome studies.

Blood Pressure↗
PubMed2001

The molecular basis of cystic fibrosis in South Africa.

The spectrum of CFTR mutations in three South African populations is presented. To date. a total of 192 white patients (384 chromosomes) with confirmed CF have been tested. deltaF508 accounts for 76% of the CF chromosomes in this group, with 3272-26A-->G, 394delTT and G542X occurring at the following frequencies: 4, 3.6 and 1.3%, respectively. A further 11 mutations account for 6% of CF chromosomes. A total of 91% of the CF-causing mutations can now be detected in the South African white population. Haplotype analysis suggests a founder effect in South Africans of European origin for the two common CFTR mutations, 3272-26A-->G and 394delTT. The diagnosis of CF has been confirmed in 14 coloured and 12 black CF patients. In the coloured population, both the deltaF508 and 3120 + 1G-->A mutations occur at appreciable frequencies of 43 and 29%, respectively. In the black population, the most common CF-causing mutation, the 3120 + 1G-->A mutation, occurs at an estimated frequency of 46%. Four other mutations have been detected, resulting in the identification of a total of 62.5% of mutations in this population.

Cystic Fibrosis↗
PubMed2001

SOOF lift and lateral retinacular canthoplasty.

Eyelid rejuvenation surgery may be slowly shifting from pure fat removal techniques to those that preserve and reposition the periorbital fat. The traditional subciliary incision blepharoplasty was fraught with minor and major complications, and while the transconjunctival blepharoplasty afforded lower morbidity, its inability to address all aspects of periorbital rejuvenation eventually limited its popularity. Coincidentally, a search by aesthetic surgeons was on for better techniques to lift the midface, soften the nasolabial fold, and efface the tear trough deformity. In an effort to avoid the lid malposition complications that often accompany transblepharoplasty cheek lifting, innovative canthoplasty and canthopexy techniques were developed, which paved the way for a safe return to subciliary blepharoplasty surgery. Effacement of the tear trough deformity, now considered to be a major determinant in successful periorbital rejuvenation, could be achieved through fat repositioning and without the use of alloplastic implants or free fat grafts, thus improving reliability while minimizing complications. By preserving periorbital fat the hollowed out orbit, often seen after traditional blepharoplasty, could be avoided. The technique described in this article is a composite of several previously published approaches, is simple for the experienced blepharoplasty surgeon to master, and has been used with safety and reliability over the past 18 months in the senior author's practice.

Adipose Tissue↗
PubMed2001

Transdermal fentanyl for pain relief in a paediatric palliative care population.

This multicentre, observational study examined the efficacy of the therapeutic transdermal fentanyl system (TTS-fentanyl) in children requiring opioids for pain in life-threatening disease. Forty-one children receiving oral morphine (median dose 60 mg/day) transferred to transdermal fentanyl (median dose 25 micrograms/h according with the manufacturer's dose conversion guidelines). Twenty-six children completed the 15-day treatment phase, seven died due to disease progression and eight were withdrawn because of adverse events, inadequate analgesia or a change to parenteral opioids. After 15 days, the median fentanyl dose was 75 micrograms/h (range 25-250). No serious adverse events were attributed to fentanyl. There was a trend toward improved side-effects and convenience with fentanyl. Twenty-three of 26 parents (three missing) and 25 of 26 investigators considered transdermal fentanyl to be better than previous treatment. For all records available (at 15 days or on withdrawal if earlier), 75% (27/36) reported that fentanyl treatment was 'good' or 'very good'. The findings suggest that transdermal fentanyl is both effective and acceptable for children and their families.

Administration, Cutaneous↗
PubMed2000

Buried charged surface in proteins.

BACKGROUND: The traditional picture of charged amino acids in globular proteins is that they are almost exclusively on the outside exposed to the solvent. Buried charges, when they do occur, are assumed to play an essential role in catalysis and ligand binding, or in stabilizing structure as, for instance, helix caps. RESULTS: By analyzing the amount and distribution of buried charged surface and charges in proteins over a broad range of protein sizes, we show that buried charge is much more common than is generally believed. We also show that the amount of buried charge rises with protein size in a manner which differs from other types of surfaces, especially aromatic and polar uncharged surfaces. In large proteins such as hemocyanin, 35% of all charges are greater than 75% buried. Furthermore, at all sizes few charged groups are fully exposed. As an experimental test, we show that replacement of the buried D178 of muconate lactonizing enzyme by N stabilizes the enzyme by 4.2 degrees C without any change in crystallographic structure. In addition, free energy calculations of stability support the experimental results. CONCLUSIONS: Nature may use charge burial to reduce protein stability; not all buried charges are fully stabilized by a prearranged protein environment. Consistent with this view, thermophilic proteins often have less buried charge. Modifying the amount of buried charge at carefully chosen sites may thus provide a general route for changing the thermophilicity or psychrophilicity of proteins.

Amino Acid Substitution↗
PubMed2000

Catalytically important ionizations along the reaction pathway of yeast pyrophosphatase.

Five catalytic functions of yeast inorganic pyrophosphatase were measured over wide pH ranges: steady-state PP(i) hydrolysis (pH 4. 8-10) and synthesis (6.3-9.3), phosphate-water oxygen exchange (pH 4. 8-9.3), equilibrium formation of enzyme-bound PP(i) (pH 4.8-9.3), and Mg(2+) binding (pH 5.5-9.3). These data confirmed that enzyme-PP(i) intermediate undergoes isomerization in the reaction cycle and allowed estimation of the microscopic rate constant for chemical bond breakage and the macroscopic rate constant for PP(i) release. The isomerization was found to decrease the pK(a) of the essential group in the enzyme-PP(i) intermediate, presumably nucleophilic water, from >7 to 5.85. Protonation of the isomerized enzyme-PP(i) intermediate decelerates PP(i) hydrolysis but accelerates PP(i) release by affecting the back isomerization. The binding of two Mg(2+) ions to free enzyme requires about five basic groups with a mean pK(a) of 6.3. An acidic group with a pK(a) approximately 9 is modulatory in PP(i) hydrolysis and metal ion binding, suggesting that this group maintains overall enzyme structure rather than being directly involved in catalysis.

Binding Sites↗
PubMed2000

New High-Resolution Analysis of the 3nu(3) and 2nu(1) + nu(3) Bands of Nitrogen Dioxide (NO(2)) by Fourier Transform Spectroscopy.

Using new high-resolution Fourier transform spectra recorded at the University of Denver in the 2-µm region, a new and more extended analysis of the 2nu(1) + nu(3) and 3nu(3) bands of nitrogen dioxide, located at 4179.9374 and 4754.2039 cm(-1), respectively, has been performed. The spin-rotation energy levels were satisfactorily reproduced using a theoretical model that takes into account both the Coriolis interactions between the spin-rotation energy levels of the (201) vibrational "bright" state with those of the (220) "dark" state. The interactions between the (003) bright state with the (022) dark state were similarly treated. The spin-rotation resonances within each of the NO(2) vibrational states were also taken into account. The precise vibrational energies and the rotational, spin-rotational, and coupling constants were obtained for the two dyads {(220), (201)} and {(022), (003)} of the (14)N(16)O(2) interacting states. From the experimental line intensities of the 2nu(1) + nu(3) and 3nu(3) bands, a determination of their vibrational transition moment constants was performed. A comprehensive list of line positions and line intensities of the {2nu(1) + 2nu(2), 2nu(1) + nu(3)} and the {2nu(2) + 2nu(3), 3nu(3)} interacting bands of (14)N(16)O(2) was generated. In addition, assuming the harmonic approximation and using the Hamiltonian constants derived in this work and in previous studies (A. Perrin, J.-M. Flaud, A. Goldman, C. Camy-Peyret, W. J. Lafferty, Ph. Arcas, and C. P. Rinsland, J. Quant. Spectrosc. Radiat. Transfer 60, 839-850 (1998)), we have generated synthetic spectra for the {(022), (003)}-{(040), (021), (002)} hot bands at 6.3 µm and for the {(220), (201)}-{(100), (020), (001)} hot bands at 3.5 µm, which are in good agreement with the observed spectra. Copyright 2000 Academic Press.

Journal Article↗
PubMed2000

Evidence that a dodecamer duplication in the gene HOPA in Xq13 is not associated with mental retardation.

A recent study suggested that a dodecamer duplication in exon 42 of the HOPA gene in Xq13 may be a significant factor in the etiology of X-linked mental retardation. In an effort to investigate this possibility, we determined the incidence of the dodecamer duplication in cohorts of non-fragile X males with mental retardation from three countries, cohorts of fragile X males from two countries, 43 probands from families with X-linked mental retardation and control cohorts from three countries. The duplication was found in 3.6-4.0% of male patients from two non-fragile X groups (Italy and South Carolina), in 1.2% from another non-fragile X group (South Africa), but in no male patients from families with X-linked mental retardation (South Carolina). The dodecamer duplication was also found in several white males with fragile X syndrome from France (5%) and South Africa (22.2%). Additionally, the duplication was found in 1.5% of South Carolinian newborn males, 2.5% South Carolinian male college students, 5% Italian male controls and 4.5% of the white South African controls. None of the black South African non-fragile X individuals with mental retardation, the fragile X or the control samples tested carried the duplication, suggesting that the duplication is rare in the black South African population. The incidence of the duplication was not significantly different between any of the groups in the study. Therefore, results of our studies in four different populations do not corroborate the findings of the previous study, and indicate that the HOPA dodecamer duplication does not convey an increased susceptibility to mental retardation.

Adult↗
PubMed2000

Reply to Schulkin.

Explore the source record for details and available documents.

Journal Article↗
PubMed2000

Production and purification of recombinant human alpha 2C2 adrenergic receptor using Saccharomyces cerevisiae.

The objective is to generate milligram quantities of recombinant human alpha 2C2 adrenergic receptor for X-ray crystallographic studies. It has been cloned in Saccharomyces cerevisiae, and the production level is at best about 13 pmol/mg of membrane protein, as estimated by radio-ligand binding assay. The receptor is solubilized with sucrose monolaurate followed by immunoaffinity purification and reconstitution into phospholipid vesicles. The efficiency of solubilization and immuno-purification are 60% and 91%, respectively.

Humans↗
PubMed2000

Resistance of very young mice to inhaled allergen sensitization is overcome by coexposure to an air-pollutant aerosol.

The role of air pollution in the initiation of asthma is controversial. We sought to model the potential effects of air pollution on immune responses to inhaled allergens in developing lungs by using very young mice. Neonatal mice were repeatedly exposed to aerosolized ovalbumin (OVA; 3% in phosphate-buffered saline for 10 min/d, from Days 5 to 15 of age). Some mice were also exposed to leachate of residual oil fly ash (ROFA-s), a surrogate for ambient air particles, for 30 min, on Days 6, 8, and 10 of age). Repeated exposure of very young mice to allergen alone (OVA) or pollutant alone (ROFA-s) had no effect on airway hyperresponsiveness (AHR, measured as enhanced pause (Penh) with noninvasive plethysmography at Day 16 of age), and did not cause inflammation or OVA-specific antibody production. Similar exposures of adult mice to either OVA alone or to OVA + ROFA-s did result in AHR, without evidence of enhancement by combined exposure. In contrast, very young mice exposed to both OVA and ROFA-s showed significantly increased AHR (e.g., Penh with 50 mg/ml methacholine for OVA + ROFA-s versus OVA alone = 2.6 +/- 0.4 [mean +/- SE], versus 1.2 +/- 0.1; p < 0.01, n >/= 15), and produced OVA-specific IgE and IgG upon allergen challenge a week later. Immunostaining of airways taken from mice at Day 11 showed a marked increase in Ia(+) cells after OVA + ROFA-s exposure. We conclude that exposure to pollutant aerosols can disrupt normal resistance to sensitization to inhaled allergens, and can thereby promote development of airway hypersensitivity in this neonatal/juvenile mouse model.

Administration, Inhalation↗
PubMed1999

Reciprocal effects of substitutions at the subunit interfaces in hexameric pyrophosphatase of Escherichia coli. Dimeric and monomeric forms of the enzyme.

A homohexameric molecule of Escherichia coli pyrophosphatase is arranged as a dimer of trimers, with an active site present in each of its six monomers. Earlier we reported that substitution of His(136) and His(140) in the intertrimeric subunit interface splits the molecule into active trimers (Velichko, I. S., Mikalahti, K., Kasho, V. N., Dudarenkov, V. Y., Hyytiä, T., Goldman, A., Cooperman, B. S., Lahti, R., and Baykov, A. A. (1998) Biochemistry 37, 734-740). Here we demonstrate that additional substitutions of Tyr(77) and Gln(80) in the intratrimeric interface give rise to moderately active dimers or virtually inactive monomers, depending on pH, temperature, and Mg(2+) concentration. Successive dissociation of the hexamer into trimers, dimers, and monomers progressively decreases the catalytic efficiency (by 10(6)-fold in total), and conversion of a trimer into dimer decreases the affinity of one of the essential Mg(2+)-binding sites/monomer. Disruptive substitutions predominantly in the intratrimeric interface stabilize the intertrimeric interface and vice versa, suggesting that the optimal intratrimeric interaction is not compatible with the optimal intertrimeric interaction. Because of the resulting "conformational strain," hexameric wild-type structure appears to be preformed to bind substrate. A hexameric triple variant substituted at Tyr(77), Gln(80), and His(136) exhibits positive cooperativity in catalysis, consistent with this model.

Amino Acid Substitution↗
PubMed1999

Pyruvate formate lyase is structurally homologous to type I ribonucleotide reductase.

BACKGROUND: Pyruvate formate lyase (PFL) catalyses a key step in Escherichia coli anaerobic glycolysis by converting pyruvate and CoA to formate and acetylCoA. The PFL mechanism involves an unusual radical cleavage of pyruvate, involving an essential C alpha radical of Gly734 and two cysteine residues, Cys418 and Cys419, which may form thiyl radicals required for catalysis. We undertook this study to understand the structural basis for catalysis. RESULTS: The first structure of a fragment of PFL (residues 1-624) at 2.8 A resolution shows an unusual barrel-like structure, with a catalytic beta finger carrying Cys418 and Cys419 inserted into the centre of the barrel. Several residues near the active-site cysteines can be ascribed roles in the catalytic mechanism: Arg176 and Arg435 are positioned near Cys419 and may bind pyruvate/formate and Trp333 partially buries Cys418. Both cysteine residues are accessible to each other owing to their cis relationship at the tip of the beta finger. Finally, two clefts that may serve as binding sites for CoA and pyruvate have been identified. CONCLUSIONS: PFL has striking structural homology to the aerobic ribonucleotide reductase (RNR): the superposition of PFL and RNR includes eight of the ten strands in the unusual RNR alpha/beta barrel as well as the beta finger, which carries key catalytic residues in both enzymes. This provides the first structural proof that RNRs and PFLs are related by divergent evolution from a common ancestor.

Acetyltransferases↗