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E Arriaga

Publications and source records attributed to E Arriaga.

17 recordsLinked to original sources
PubMed2000

Single-cell analysis avoids sample processing bias.

Microscale separation tools such as capillary chromatography and capillary electrophoresis (CE) allow the study of metabolism in individual cells. In this work, we demonstrate that single-cell analysis describes metabolism more accurately than analysis of cellular extracts. We incubated HT29 cells (human colon adenocarcinoma) with a fluorescently labeled metabolic probe. This disaccharide, LacNAc, was labeled with a fluorescent dye, tetramethylrhodamine (TMR). The probe was taken up by the cells and metabolized to a number of products that retained the fluorescent label. We then split the cells into two batches. A cellular extract was prepared from one batch and analyzed by CE with laser-induced fluorescence (LIF) detection. The cells from the second batch were used for single-cell analysis by CE-LIF. Separation and detection conditions were identical for extract and single-cell analyses. We found that the electropherogram obtained by averaging the results from a number of single cells differed significantly from the cell extract electropherogram. Differences were due to sample processing during extract preparation. Disruption of the cells liberated enzymes that were compartmentalized within the cell, which allowed non-metabolic reactions to proceed. The accumulation of these non-metabolic products introduced a bias in the cell extract assay. During single-cell analysis, cells were lysed inside the capillary and the separation voltage was applied immediately to separate the enzymes from their substrates and prevent non-metabolic reactions. This paper is the first to report that CE analysis of single cells provides more accurate metabolic information than the CE analysis of a cellular extract.

Electrophoresis, Capillary↗
PubMed1999

Correlating cell cycle with metabolism in single cells: combination of image and metabolic cytometry.

BACKGROUND: We coin two terms: First, chemical cytometry describes the use of high-sensitivity chemical analysis techniques to study single cells. Second, metabolic cytometry is a form of chemical cytometry that monitors a cascade of biosynthetic and biodegradation products generated in a single cell. In this paper, we describe the combination of metabolic cytometry with image cytometry to correlate oligosaccharide metabolic activity with cell cycle. We use this technique to measure DNA ploidy, the uptake of a fluorescent disaccharide, and the amount of metabolic products in a single cell. METHODS: A colon adenocarcinoma cell line (HT29) was incubated with a fluorescent disaccharide, which was taken up by the cells and converted into a series of biosynthetic and biodegradation products. The cells were also treated with YOYO-3 and Hoechst 33342. The YOYO-3 signal was used as a live-dead assay, while the Hoechst 33342 signal was used to estimate the ploidy of live cells by fluorescence image cytometry. After ploidy analysis, a cell was injected into a fused-silica capillary, where the cell was lysed. Fluorescent metabolic products were then separated by capillary electrophoresis and detected by laser-induced fluorescence. RESULTS: Substrate uptake measured with metabolic cytometry gave rise to results similar to those measured by use of laser scanning confocal microscopy. The DNA ploidy histogram obtained with our simple image cytometry technique was similar to that obtained using flow cytometry. The cells in the G(1) phase did not show any biosynthetic activity in respect to the substrate. Several groups of cells with unique biosynthetic patterns were distinguished within G(2)/M cells. CONCLUSIONS: This is the first report that combined metabolic and image cytometry to correlate formation of metabolic products with cell cycle. A complete enzymatic cascade is monitored on a cell-by-cell basis and correlated with cell cycle.

Biodegradation, Environmental↗
PubMed1999

Single cell studies of enzymatic hydrolysis of a tetramethylrhodamine labeled triglucoside in yeast.

Several hundred molecules of enzyme reaction products were detected in a single spheroplast from yeast cells incubated with a tetramethylrhodamine (TMR) labeled triglucoside, alpha-d-Glc(1-->2)alpha-d-Glc(1-->3)alpha-d-Glc-O(CH2)8CONHCH2- CH2NH- COTMR. Product detection was accomplished using capillary electrophoresis and laser induced fluorescence following the introduction of a single spheroplast into the separation capillary. The in vivo enzymatic hydrolysis of the TMR-trisaccharide involves at least two enzymes, limited by processing alpha-glucosidase I, producing TMR-disaccharide, TMR-monosaccharide, and the free TMR-linking arm. Hydrolysis was reduced by preincubation of the cells with the processing enzyme inhibitor castanospermine. Confocal laser scanning microscopy studies confirmed the uptake and internalization of fluorescent substrate. This single cell analysis methodology can be applied for the in vivo assay of any enzyme with a fluorescent substrate.

Cytological Techniques↗
PubMed1998

Sodium dodecyl sulfate-capillary electrophoresis of proteins in a sieving matrix utilizing two-spectral channel laser-induced fluorescence detection.

We report a method for protein labeling, separation by capillary electrophoresis in a polymer sieving matrix, and detection by laser-induced fluorescence. Different dyes are used to label standard and sample proteins. A two-spectral channel detector resolves fluorescence from the sample and standards. Comparison of the migration time of the sample and standards permits the precise determination of molecular weight, irrespective of variations in run-to-run migration times.

Conalbumin↗
PubMed1996

Pseudo-coulometric loading in capillary electrophoresis DNA sequencing.

While injection volumes in capillary electrophoresis are typically in the nanoliter range, it is difficult to physically prepare and manipulate samples much smaller than a microliter. As a result, only a small fraction of the analyte contained with the sample volume is transferred to the capillary. This problem is particularly acute in DNA sequencing applications, where on-column stacking is difficult and where the sequencing sample is relatively expensive to prepare. We report a method that transfers 75% of the DNA contained within a 3 microliters sample onto a capillary for DNA sequencing. This method relies on the use of very low ionic strength formamide to resuspend the DNA after an ethanol precipitation. The use of low ionic strength formamide achieves two tasks. First, it produces a very high resistance sample, which increases the voltage drop across the sample and decreases the field across the capillary. This electric field manipulation ensures that DNA fragments do not migrate down the capillary during the loading process, allowing long injection periods without excessive band-broadening. Second, the low ionic strength of the formamide increases the transference number of the DNA; more of the current passing through the injection tip of the capillary is carried by DNA fragments. In the limit of complete elimination of impurity ions from the loading solvent, current passing through the sample is carried only by DNA fragments and loading becomes a coulometric process.

DNA, Single-Stranded↗
PubMed1995

Progression of cardiac potassium current modification after brief exposure to reactive oxygen.

We reported previously that singlet oxygen (1O2), generated by illuminating the photosensitizer rose bengal (RB), suppressed the delayed rectifier potassium current (IK) in single frog atrial cells. Considering the brief lifetime of 1O2, one might expect IK modification to reach a steady-state soon after a brief exposure to RB-generated 1O2. Here we report that, contrary to expectations, tens of seconds can be required for IK to reach a new steady-state. We will use the term "progression" to refer to the component of current modification which occurs after cessation of illumination. To gain insight into the mechanism of progression, we investigated how its time course and magnitude were affected by (1) membrane potential during and following RB illumination, and (2) the level of IK activation during illumination. We found that conditions which favored the open state of the potassium channel also favored progression, increasing both its time course and magnitude. Illumination while IK was activated produced significant progression having a very slow time course (tens of seconds). By comparison, illumination when IK was not activated produced no progression: IK modification was completed during the 2 s illumination period. These findings suggest progression results from the kinetics of potassium channel state transitions rather than from a long-lived reactive intermediate produced during the initial 1O2 exposure.

Animals↗
PubMed1994

Separation of fragments up to 570 bases in length by use of 6% T non-cross-linked polyacrylamide for DNA sequencing in capillary electrophoresis.

Non-cross-linked polyacrylamide is a very convenient medium for the separation of DNA sequencing fragments in capillary electrophoresis. We demonstrate DNA sequencing with this matrix at an electric field of 200 V/cm and at room temperature. Resolution is observed to decrease exponentially with fragment length. Fragments 570 bases in length generate a resolution of 0.5, which is adequate for sequence identification.

Cross-Linking Reagents↗
PubMed1994

High-speed and high-accuracy DNA sequencing by capillary gel electrophoresis in a simple, low cost instrument. Two-color peak-height encoded sequencing at 40 degrees C.

A low-cost DNA sequencer was constructed based on a single helium-neon laser. The two-color peak-height encoded sequencing protocol, based on the use of T7 DNA polymerase in a manganese buffer, was used to generate samples. Two termination reactions were performed. In the first, a TAMRA (applied Biosystems)-labeled primer was extended in the presence of ddATP and ddCTP. The amounts of dideoxynucleotides were adjusted to produce a 3:1 peak height ratio. Similarly, a ROX (Applied Biosystems)-labeled primer was extended in the presence of ddGTP and ddTTP; the amounts of dideoxynucleotides was adjusted to produce a 3:1 peak height ratio. The pooled fragments were separated on a 4% T LongRanger gel operated at 39 degrees C. Over 500 bases of sequence were generated in 50 min.

Capillary Action↗
PubMed1994

Activation energy of single-stranded DNA moving through cross-linked polyacrylamide gels at 300 V/cm. Effect of temperature on sequencing rate in high-electric-field capillary gel electrophoresis.

In DNA sequencing, single-stranded DNA fragments are separated by gel electrophoresis. This separation is based on a sieving mechanism where DNA fragments are retarded as they pass through pores in the gel. In this paper, we present the mobility of DNA sequencing fragments as a function of temperature; mobility is determined in 4% T LongRanger gels at an electric field of 300 V/cm. The temperature dependence is compared with the predictions of the biased reptation model. The model predicts that the fragment length for the onset of biased reptation with stretching increases with the square of temperature; the data show that the onset of biased reptation with stretching decreases with temperature. Biased reptation fails to model accurately the temperature dependence of mobility. We analyzed the data and extracted the activation energy for passage of sequencing fragments through the gel. For fragments containing less than ca. 200 bases, the activation energy increases linearly with the number of bases at a rate of 25 J/mol per base; for longer fragments, the activation energy increases at a rate of 6.5 J/mol per base. This transition in the activation energy presumably reflects a change in conformation of the DNA fragments; small fragments exist in a random coil configuration and larger fragments migrate in an elongated configuration.

Chemical Phenomena↗
PubMed1994

Properties of cardiac I(leak) induced by photosensitizer-generated reactive oxygen.

We reported previously that photomodification of single frog cardiac cells by Rose Bengal induces a time-independent current, designated I(leak)++, having a linear current-voltage (I/V) relationship. The purpose of the present study is to better characterize the properties of I(leak)++. Initially, I(leak)++ has a reversal potential (ER) near -70 mV, but with time, ER shifts toward a final value near 0 mV. This shift in ER is accompanied by a marked increase in conductance (slope of I/V relationship). Evidence is presented that the depolarizing shift in ER with time during photomodification results from a loss of membrane selectivity allowing sodium to make an increasing contribution to I(leak)++. Potassium also contributes to I(leak)++, as indicated by marked depolarizing shifts in ER following replacement of intracellular potassium with either cesium or tetraethylammonium. Since these results occur in calcium-free external media, the depolarizing shifts in ER and increased conductance are not related to activation of a calcium-dependent nonselective cation channel. However, I(leak) does have some properties similar to nonselective cation currents recently reported to be activated by membrane breakdown products such as arachidonic acid and lysophosphoglycerides.

Animals↗
PubMed1994

Membrane ionic current photomodification by rose bengal and menadione: role of singlet oxygen.

Photosensitized modification of ionic leak current and potassium current was studied in frog cardiac atrial cells using whole cell patch clamp techniques. Rose bengal (RB) and menadione (MQ) were used as photosensitizers. Separate photophysical studies of the photosensitizers in deuterium oxide solution demonstrated that MQ did not produce singlet oxygen as evidenced by the lack of luminescence at 1270 nm, whereas RB was an efficient singlet oxygen generator. Both photosensitizers sensitized block of potassium current in atrial cells, and both sensitized an increase of ionic leak current. However, when photosensitizer concentrations and illumination intensities were adjusted to match the rate of block of potassium current by the two photosensitizers, there were dramatic differences in leak current increase, both quantitatively and qualitatively. Menadione sensitized a much slower increase in leak current than did RB. Further, the leak current sensitized by MQ had a more positive reversal potential than that sensitized by RB, suggesting a less potassium-selective leak current pathway. The results suggest that, while the effects of singlet oxygen and non-singlet oxygen modification of cell membranes may be similar, there may also be significant differences in the resulting membrane permeabilities. The results also demonstrate that MQ and RB may be useful agents to study the role of singlet oxygen versus non-singlet oxygen modification of biological systems.

Animals↗
PubMed1993

Membrane potential can influence the rate of membrane photomodification.

Though cellular photomodification has been shown to change cellular resting membrane potential, an effect of membrane potential on the rate of photomodification has never been reported. Here we demonstrate that the rate of photomodification of potassium channels in frog atrial cells is voltage dependent. The rate of potassium channel photomodification using negatively charged Rose Bengal as the photosensitizer is about 2.5 times greater at the resting membrane potential of -70 mV compared to +40 mV. Similar results are obtained using the positively charged photosensitizer methylene blue. On the other hand, the rate of photomodified increase of leak current in the same cells does not significantly change in this voltage range with Rose Bengal as photosensitizer, but demonstrates a voltage dependence like that of potassium current when methylene blue is the photosensitizer. These observations cannot be explained based on voltage-dependent partitioning of the sensitizer, as similar effects on potassium current were obtained using either a positively charged or negatively charged sensitizer.

Animals↗
PubMedDate not supplied

Thinner abuse alters optokinetic nystagmus parameters.

BACKGROUND: Deliberate self-inhalation of solvents such as thinner is a recognized problem in underdeveloped countries, with chronic abuse resulting in neurological impairment. In this article, we use electronystagmography (ENG) to study optokinetic nystagmus abnormalities (OKN) that may be induced by thinner consumption. METHODS: Twenty-five patients exposed to thinner for 5-20 years, in an irregular fashion of consumption, were recruited from a toxicologic center. Twenty-five control subjects were invited to participate as volunteers matched by age (+/-2 years) and gender. At the time of evaluation, all had abstained from intoxicants for at least 4 weeks. ENG recordings were performed by clinicians masked to the patient's group. Clockwise and counterclockwise stimulation were performed at 20 and 40 degrees /sec. RESULTS: None of the patients showed spontaneous nystagmus during the test period. Differences between thinner abusers and controls on clockwise and counterclockwise OKN on number of beats of nystagmus elicited on the 40 degrees /sec velocity were identified. The thinner abusers group showed a lesser number of nystagmus (p level was 0.02 and 0.005, respectively). CONCLUSIONS: The present results confirm the sensibility of OKN as an early marker of solvent abuse. These results were obtained in middle-term chronic exposure to solvent mixtures and are in favor of both cortical and brainstem dysfunction.

Adolescent↗
PubMedDate not supplied

Recording esophageal heart sounds with a catheter-tip contact microphone.

The use of a catheter-tip microphone is described for the detection of heart sounds in the esophagus. The intensity and quality of the sounds differ from those detected on the precordium. Individual valve and breath sounds can be attenuated by choice of the location of the microphone in the esophagus. The acoustic shielding provided by the thorax allows presentation of heart sounds with little difficulty from acoustic feedback.

Animals↗