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A Finkelstein

Publications and source records attributed to A Finkelstein.

At least 55 records · Page 3Linked to original sources
PubMed1994

Structure-function relationships in diphtheria toxin channels: III. Residues which affect the cis pH dependence of channel conductance.

The conductance of channels formed by diphtheria toxin (DT) in lipid bilayer membrane depends strongly on pH. We have previously shown that a 61 amino acid region of the protein, denoted TH8-9, is sufficient to form channels having the same pH-dependent conductance properties as those of whole toxin channels. One residue in this region, Aspartate 352, is responsible for all the dependence of single channel conductance on trans pH, whereas another, Glutamate 349, has no effect. Here, we report that of the seven remaining charged residues in the TH8-9 region, mutations altering the charge on H322, H323, H372, and R377 have minimal effects on single channel conductance; mutations of Glutamates 326, 327, or 362, however, significantly affect single channel conductance as well as its dependence on cis pH. Moreover, Glutamate 362 is titratable from both the cis and trans sides of the membrane, suggesting that this residue lies within the channel; it is more accessible, however, to cis than to trans protons. These results are consistent with the membrane-spanning topology previously proposed for the TH8-9 region, and suggest a geometric model for the DT channel.

Aspartic Acid↗
PubMed1993

Structure/function analysis of the transmembrane domain of DAB389-interleukin-2, an interleukin-2 receptor-targeted fusion toxin. The amphipathic helical region of the transmembrane domain is essential for the efficient delivery of the catalytic domain to the cytosol of target cells.

Cassette and deletion mutagenesis were used to analyze the function of the amphipathic alpha-helices in the transmembrane domain of DAB389-interleukin-2 (IL-2), a fusion protein which is targeted to the interleukin-2 receptor. We demonstrate that the in-frame deletion of 60 amino acids, from Asn204 to Glu263 in DAB389-IL-2, results in complete loss of cytotoxic activity, whereas when the amphipathic regions from Asp208 to Ser220 and Ala244 to His258 are replaced with idealized amphipathic helices composed of repeating Glu, Lys, and Leu residues, the mutant fusion toxin has low but detectable activity. DAB389-IL-2 and both variants form channels in artificial phospholipid bilayers with conductances identical to those formed by diphtheria toxin. Both mutant fusion toxins bind to the high affinity IL-2 receptor with affinities similar to that of DAB389-IL-2. The fact that these mutants have markedly reduced or absent cytotoxic activity, but possess "wild type" catalytic activity, binding affinities, and channel conductances, suggests the existence of a step in the intoxication pathway, defective in the mutants, which occurs after DAB389-IL-2 binds to the IL-2 receptor. It is unknown whether this step occurs prior or subsequent to channel formation, but it is essential for the efficient delivery of the ADP-ribosyltransferase from DAB389-IL-2 to the cytosol of target cells.

Amino Acid Sequence↗
PubMed1993

Production of human RAP30 and RAP74 in bacterial cells.

RAP30 and RAP74 are subunits of RAP30/74 (TFIIF), a general initiation and elongation factor for transcription by RNA polymerase II. Complementary DNA (cDNA) clones have previously been reported encoding human RAP30 and RAP74. Here we report expression of these cDNAs using a T7 RNA polymerase system in Escherichia coli. Production of human RAP30 was very efficient using the expression vector pET11d. RAP30 accumulated within inclusion bodies and was solubilized using guanidine hydrochloride. After removal of the denaturant, RAP30 was soluble and active in accurate transcription. Approximately 44 mg of highly purified and soluble RAP30 was obtained from a 1-liter culture of cells. Production of RAP74 was more problematic, because a mixture of full length RAP74 and RAP74 fragments was produced in E. coli. Most RAP74 fragments were shortened by deletion of the COOH-terminus of the protein and probably resulted from premature translation termination. RAP74 was most successfully produced using a pET23d construct, in which the RAP74 peptide was fused to a short polyhistidine stretch at its COOH-terminus. Addition of the polyhistidine sequence allowed purification using a Ni2+ affinity resin. Full length RAP74 carrying this polyhistidine extension was purified in a single step by Ni2+ affinity chromatography in 4 M urea; the yield of RAP74 was approximately 3 mg from a 1-liter culture of cells. RAP74 derivatized with a polyhistidine stretch at its NH2-terminus, on the other hand, remained contaminated with RAP74 fragments after Ni2+ affinity chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Effect of surgical mask position on bacterial contamination of the operative field.

The influence of surgical mask usage on bacterial contamination of the operative field was studied during 30 cardiac catheterization procedures. Mask position was varied during each procedure according to a predesigned random table. The number of bacterial colonies recoverable when no mask was worn was significantly higher than that detected when a full mask was worn (P < 0.002). Shedding of Staphylococcus epidermidis was greater when no mask was worn (mean 5.2 colonies 10 min-1) than shedding with full mask (mean 2.7 colonies 10 min-1; P < 0.004). Although mask placement below the nose was associated with higher mean colony counts than that above the nose, these differences were not statistically significant.

Bacteria↗
PubMed1992

A cDNA encoding RAP74, a general initiation factor for transcription by RNA polymerase II.

RAP30/74 (also known as TFIIF, beta gamma and FC is one of several general factors required for initiation by RNA polymerase II. The small RAP30 subunit of RAP30/74 binds directly to polymerase and appears structurally and functionally homologous to bacterial sigma factors in their RNA polymerase-binding region. RAP30/74 or recombinant RAP30 suppresses nonspecific binding of RNA polymerase II to DNA and is required for RNA polymerase II to assemble stably into a preinitiation complex containing promoter DNA and the general factors TFIID, TFIIA and TFIIB; both RAP30 and RAP74 are physical components of the preinitiation complex. A complementary DNA encoding human RAP30 has been isolated, and here we report the isolation of a cDNA encoding human RAP74. RAP30 and RAP74 produced in Escherichia coli can be used in place of natural human RAP30/74 to direct accurate transcription initiation by RNA polymerase II in vitro.

Amino Acid Sequence↗
PubMed1992

Locating a residue in the diphtheria toxin channel.

We are studying structure-function relationships in the Diphtheria Toxin (DT) channel using a combination of site-directed mutagenesis and electrophysiology in artificial lipid bilayers. We made site-directed mutations of charged residues in the toxin's channel-forming B fragment, and after expressing the mutant proteins in Escherichia coli, we analyzed the single channels they formed in lipid bilayers. Changing aspartate 352, which is located in a short hydrophilic loop separating two hydrophobic stretches, to asparagine or lysine dramatically reduces the single-channel conductance of the pore at pH 5.3 cis, 7.2 trans (5.3/7.2). Lowering the pH on both sides of the membrane essentially eliminates the difference between wild-type and D352N; this finding is consistent with the idea that an aspartate with a (protonated) neutral side-chain and the always neutral asparagine have similar electrostatic influences on permeant ions. Using a high concentration of permeant buffer to clamp the pH of the cis compartment and the pore, and varying the pH on the trans side, we have located D352 at or near the trans compartment. We further find that D352N channels, in contrast to wild-type, display conductances independent of trans pH. This observation allows us to determine the titration curve of aspartate 352 in the wild-type toxin, establishing its pKa at approximately 5.5.

Amino Acid Sequence↗
PubMed1992

Combined effect of captopril and aspirin in renal hemodynamics in elderly patients with congestive heart failure.

Captopril and aspirin have been claimed to adversely affect renal function. This study was designed to evaluate the safety of concomitant administration of both drugs in patients with moderate to severe congestive heart failure (CHF). The study group consisted of 10 patients with a mean age of 77.6 +/- 4.4 years and a mean New York Heart Association functional class of 2.6 +/- 0.5. Captopril was administered in a rapidly escalating dose regimen over a 4-day period to a maximum dose of 75 mg/day. Aspirin 0.25 g/day was added from day 5 on. Renal plasma flow (RPF) was measured by iodohippurate scan and the creatinine clearance test (Ccr) was used as an index of glomerular filtration rate (GFR). Both Ccr and RPF remained unchanged throughout the study period; 48.9 +/- 16; 48.2 +/- 16.5; 49.4 +/- 16, and 222 +/- 67, 241 +/- 97, 237 +/- 88 ml/min, for days 0, 4, 9, respectively. Only 1 patient developed a significant decrease in Ccr following the administration of captopril. This patient had a further decrease when aspirin was added. The decrease in Ccr was accompanied by a marked reduction in filtration fraction and in mean arterial pressure. Our data suggest that the administration of aspirin to elderly patients with moderate CHF treated with captopril is relatively safe and is not associated with further deterioration in renal function.

Aged↗
PubMed1991

The effects of hormone replacement therapy in normal postmenopausal women: measurements of Doppler-derived parameters of aortic flow.

In this study the effects of hormone replacement therapy on cardiac function in healthy postmenopausal women were evaluated by Doppler echocardiography that was performed before (T1) and 2.5 months after the initiation of hormone replacement therapy (T2) in the peak estrogenic phase. The following parameters of aortic flow were measured: peak flow velocity, acceleration time, and ejection time. Additional parameters were calculated: flow velocity integral and mean acceleration. The study group included 24 postmenopausal women aged 43 to 60 years (mean 51.6 years). The control group consisted of 19 postmenopausal women aged 46 to 60 years (mean 53.5 years) who were not receiving hormone replacement therapy and who underwent the same evaluation. There were no changes in all Doppler parameters between T1 and T2 in the control group. However, in the study group there were significant increases in peak flow velocity (108.3 +/- 16.7 cm/sec at T1 vs 123 +/- 20.7 cm/sec at T2; p = 0.002), flow velocity integral (17.7 +/- 3.9 vs 21.5 +/- 4.7 cm; p = 0.0003), mean acceleration (11.5 +/- 1.9 vs 13.1 +/- 2.6 m/sec/sec; p = 0.001), and ejection time (324 +/- 37.6 vs 348.8 +/- 40.7 msec; p = 0.002). There was no change in acceleration time (94.8 +/- 6.6 vs 95 +/- 10.9 msec). These results demonstrate that estrogens increase both stroke volume and flow acceleration. The latter probably reflects a combination of enhanced inotropism and vasodilatation. We assume that the cardioprotective effect of hormone replacement therapy in postmenopausal women may be due not only to changes in lipid profile but also to direct effects of estrogens on central and peripheral hemodynamic parameters.

Adult↗
PubMed1991

Is idiopathic atrial fibrillation caused by occult thyrotoxicosis? A study of one hundred consecutive patients with atrial fibrillation.

In this study we evaluated the possible relationship between idiopathic atrial fibrillation and occult thyrotoxicosis, diagnosed by lack of response of thyroid stimulating hormone to administration of thyrotropin releasing hormone. Three groups were compared: 25 patients with idiopathic atrial fibrillation; 52 with cardiovascular atrial fibrillation; and 27 with sinus rhythm. Patients were excluded with any clinical evidence of thyrotoxicosis or with elevated serum FT4 level, as well as those with diseases or on medications known to be associated with a diminished response to administration of the releasing hormone. A flat test (lack of response) was found in only 4% of the patients. There was no significant difference among the 3 groups. Based on these data we believe that there is no relationship between idiopathic atrial fibrillation or any other type of atrial fibrillation and occult thyrotoxicosis.

Adult↗
PubMed1991

Identification of a translocated gating charge in a voltage-dependent channel. Colicin E1 channels in planar phospholipid bilayer membranes.

The availability of primary sequences for ion-conducting channels permits the development of testable models for mechanisms of voltage gating. Previous work on planar phospholipid bilayers and lipid vesicles indicates that voltage gating of colicin E1 channels involves translocation of peptide segments of the molecule into and across the membrane. Here we identify histidine residue 440 as a gating charge associated with this translocation. Using site-directed mutagenesis to convert the positively charged His440 to a neutral cysteine, we find that the voltage dependence for turn-off of channels formed by this mutant at position 440 is less steep than that for wild-type channels; the magnitude of the change in voltage dependence is consistent with residue 440 moving from the trans to the cis side of the membrane in association with channel closure. The effect of trans pH changes on the ion selectivity of channels formed by the carboxymethylated derivative of the cysteine 440 mutant independently establishes that in the open channel state, residue 440 lies on the trans side of the membrane. On the basis of these results, we propose that the voltage-gated opening of colicin E1 channels is accompanied by the insertion into the bilayer of a helical hairpin loop extending from residue 420 to residue 459, and that voltage-gated closing is associated with the extrusion of this loop from the interior of the bilayer back to the cis side.

Base Sequence↗
PubMed1990

Alteration of the pH-dependent ion selectivity of the colicin E1 channel by site-directed mutagenesis.

Colicin E1 is a soluble, bacteriocidal protein that forms voltage-gated channels in planar lipid bilayers. The channel-forming region of the 522-amino acid protein is near the COOH terminus, and contains a 35-amino acid hydrophobic segment which is presumed to be important in interacting with the membrane. We have used site-directed mutagenesis in the region immediately upstream from the hydrophobic segment to construct several functional colicin mutants in which a wild-type residue was replaced with a cysteine. We also replaced the only naturally occurring cysteine in the molecule, Cys-505, with alanine, so that synthetically introduced cysteines could unambiguously serve as targets for chemical modification. All of the replacements reported here (at positions 449, 459, 473, 505, and some combinations) resulted in a channel that had an ion selectivity (K+ versus Cl-) identical to wild type at low pH. At higher pH, however, one of these mutations, which replaced the negatively charged aspartate at position 473 (the upstream boundary of the hydrophobic segment), resulted in a channel that was less cation-selective than was wild type. When the introduced Cys-473 was reacted with iodoacetic acid, which inserted a COOH group close to the position of the missing aspartate COOH, wild-type ion selectivity was restored, suggesting that the greater cation selectivity of the wild-type channel was directly produced by the negative charge at Asp-473. By comparing the ion selectivity of the Cys-473 mutant channel to that of the wild type as a function of the pH on the cis and trans sides of the membrane, it was possible to locate residue 473 close to the cis side. Locating in this manner the positions in the channel of particular residues places important constraints on channel model building.

Amino Acid Sequence↗
PubMed1990

Transactivation of the Rous sarcoma virus long terminal repeat promoter by Marek's disease virus.

Transient expression of chloramphenicol acetyltransferase (CAT) was used to study Marek's diseases virus (MDV)-mediated transactivation of the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter. Cotransfection experiments in primary avian cells were conducted using MDV high-molecular-weight DNA and plasmid pRSVcat. Increased CAT activity, relative to controls, was consistently observed in the presence of MDV. Enhanced CAT activity, expressed via the RSV-LTR promoter, was strictly dependent on the presence of MDV DNA or virus, suggesting that activation of the RSV-LTR promoter was due to factors expressed in MDV-infected cells. Differences in transactivation efficiency were observed between various strains and the serotypes of MDV. In particular, high- and low-passage pairs of serotype 1 MDV showed marked differences in their ability to increase CAT activity in pRSVcat-transfected cells. Attenuation of viral pathogenicity and decreased expression of some cell surface glycoproteins occur in high-passage MDV strains. Decreased transactivation ability in these same strains suggests that continuous passage in culture and attenuation may perturb a regulatory mechanism operating by transcriptional control. In addition, transactivation of the RSV-LTR promoter suggests that increased incidence of avian leukosis following vaccination by MDV may be due to MDV-mediated transactivation of endogenous ALV proviral LTR promoters. MDV-mediated transactivation was not limited to the RSV-LTR promoter. Serotype 3 MDV (HVT) efficiently transactivated the herpes simplex virus (HSV) alpha 4 (ICP4) and beta-TK promoters as well as the human cytomegalovirus (hCMV) immediate early promoter.

Animals↗
PubMed1990

Voltage-dependent block of anthrax toxin channels in planar phospholipid bilayer membranes by symmetric tetraalkylammonium ions. Effects on macroscopic conductance.

In a recent paper (Blaustein, R. O., T. M. Koehler, R. J. Collier, and A. Finkelstein, 1989. Proc. Natl. Acad. Sci. USA. 86:2209-2213) we described the general channel-forming properties of the PA65 fragment of anthrax toxin in planar phospholipid bilayer membranes. In the present paper we extend our previous studies of the permeability properties of this channel, using a series of symmetric tetraalkylammonium (TAA) ions. Our main finding is that at micromolar concentrations on either the cis (toxin-containing) or trans side of a membrane containing many (greater than 1,000) channels, these ions, ranging in size from tetramethylammonium to tetrahexylammonium, induce a voltage-dependent reduction of membrane conductance. (We attribute a similar voltage-dependent reduction of membrane conductance by millimolar concentrations of HEPES to a cationic form of this buffer present at micromolar concentrations.) In going from large negative to large positive voltages (on the TAA side) one sees that the conductance first decreases from its value in the absence of TAA, reaches a minimum, and then rises back at larger positive voltages toward the level in the absence of TAA. Our interpretation of this behavior is that these symmetric TAA ions block the cation-selective PA65 channel in a voltage-dependent manner. We postulate that there is a single site within the channel to which TAA ions can bind and thereby block the passage of the major current-carrying ion (potassium). A blocking ion is driven into the site by modest positive voltages, but is driven off the site and through the channel by larger positive voltages, thus explaining the relief of block. (In the accompanying paper [Blaustein, R. O., E. J. A. Lea, and A. Finkelstein. 1990. J. Gen. Physiol. 96:921-942] we confirm this interpretation of the data by analysis at the single-channel level.) This means that these blocking ions can pass through the channel; the permeability to tetrahexylammonium, the largest ion studied, implies that the narrowest part of the channel has a diameter of at least 11 A.

Antigens, Bacterial↗
PubMed1990

Voltage-dependent block of anthrax toxin channels in planar phospholipid bilayer membranes by symmetric tetraalkylammonium ions. Single-channel analysis.

Previous studies have shown that symmetric tetraalkylammonium ions affect, in a voltage-dependent manner, the conductance of membranes containing many channels formed by the PA65 fragment of anthrax toxin. In this paper we analyze this phenomenon at the single-channel level for tetrabutylammonium ion (Bu4N+). We find that Bu4N+ induces a flickery block of the PA65 channel when present on either side of the membrane, and this block is relieved by large positive voltages on the blocking-ion side. At high frequencies (greater than 2 kHz) we have resolved individual blocking events and measured the dwell times in the blocked and unblocked states. These dwell times have single-exponential distributions, with time constants tau b and tau u that are voltage dependent, consistent with the two-barrier, single-well potential energy diagram that we postulated in our previous paper. The fraction of time the channel spends unblocked [tau u/(tau u + tau b)] as a function of voltage is identical to the normalized conductance-voltage relation determined from macroscopic measurements of blocking, thus demonstrating that these single channels mirror the behavior seen with many (greater than 10,000) channels in the membrane. In going from large negative to large positive voltages (-100 to +160 mV) on the cis (PA65-containing) side of the membrane, one sees the mean blocked time (tau b) increase to a maximum at +60 mV and then steadily decline for voltages greater than +60 mV, thereby clearly demonstrating that Bu4N+ is driven through the channel by positive voltages on the blocking-ion side. In other words, the channel is permeable to Bu4N+. An interesting finding that emerges from analysis of the voltage dependence of mean blocked and unblocked times is that the blocking rate, with Bu4N+ present on the cis side of the membrane, plateaus at large positive cis voltages to a voltage-independent value consistent with the rate of Bu4N+ entry into the blocking site being diffusion limited.

Antigens, Bacterial↗
PubMed1990

Diffusion limitation in the block by symmetric tetraalkylammonium ions of anthrax toxin channels in planar phospholipid bilayer membranes.

Current flow through the channel formed in planar phospholipid bilayer membranes by the PA65 fragment of anthrax toxin is blocked, in a voltage-dependent manner, by tetraalkylammonium ions (at micromolar concentrations), which bind to a blocking site within the channel lumen. We have presented evidence that diffusion plays a significant role in the kinetics of blocking by tetrabutylammonium ion (Bu4N+) from the cis (toxin-containing) side of the membrane (Blaustein, R. O., E. J. A. Lea, and A. Finkelstein. 1990. J. Gen. Physiol. 96:921-942); in this paper we examine the implications and consequences of diffusion control for binding kinetics. As expected for a diffusion-affected reaction, both the entry rate constant (kcis1) of Bu4N+ from the cis solution to the blocking site and the exit rate constant (kcis-1) of Bu4N+ from the blocking site to the cis solution are reduced if the viscosity of that medium is increased by the addition of dextran. In conformity with both thermodynamics and kinetic arguments, however, the voltage-dependent equilibrium binding constant, Keq (= kcis-1/kcis1), is not altered by the dextran-induced viscosity increase of the cis solution. The entry rate constants (kcis1) for tetrapentylammonium (Pe4N+), tetrahexylammonium (Hx4N+), and tetraheptylammonium (Hp4N+) are also diffusion controlled, and all of them, including that for Bu4N+, attain a voltage-independent plateau value at large positive cis voltages consistent with diffusion limitation. Although the plateau value of kcis1 for Hx4N+ is only a factor of 3 less than that for Bu4N+, the plateau value for Hp4N+ is a factor of 35 less. This precipitous fall in value indicates, from diffusion-limitation theory, that the diameter of the channel entrance facing the cis solution is not much larger than the diameter of Hp4N+, i.e., approximately 12 A.

Antigens, Bacterial↗