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PubMed2002

Influence of chromatographic conditions on separation in comprehensive gas chromatography.

Comprehensive gas chromatography (GC x GC) is now established as a powerful technique, which offers unprecedented separation power. For complex samples, the distribution of peaks in the two-dimensional (2D) space still may need to be optimised. Since temperature (T) is a critical variable, and compounds can be shifted in relative positions on column 1 arising from temperature program rate (rT) changes, and since retention in the second dimension, D2 (2tR) is likewise affected by the prevailing T (elution temperature; Te), then any factors which alter Te will affect the extent of separation in D2. Since temperature program and carrier gas velocity rate will affect the Te of the solutes, these two factors are considered in this paper. Apart from these two parameters, results of different stationary phase choice for the second dimension column as well as the second dimension column length are reported. rT is found to have the most profound impact on the Te of solutes and will be more likely to cause an inversion of elution order if such behaviour can occur. Peak widths and 2tR increase with a decrease in Te. On the other hand, flow-rate has less impact on peak widths and 2tR although Te is affected by a change in flow-rate. Specific solute-stationary phase interactions will cause the elution order of certain solutes to be altered, and may be observed when a different stationary phase is employed as the second column, depending on the solute-stationary phase separation mechanism. Experiments conducted on different second dimension column length showed that although a longer column will lead to better separations, wrap-around may confound the separation process and may cause the solutes from sequential modulation events to co-elute. Thus a suitable second dimension column phase and length must be employed in order to obtain good separation. The factors investigated in this study will cause different extents of changes in the solute elution order and solute separations, and will affect the 2D contour presentation.

Chromatography, Gas↗
PubMed2002

Chiral separation of amino acids by capillary electrophoresis with octyl-beta-thioglucopyranoside as chiral selector.

1-S-Octyl-beta-D-thioglucopyranoside (OTG) was evaluated as a chiral selector for the separation of dansyl-amino acids by capillary electrophoresis. Enantiomeric separations of the amino acids can be accomplished by judiciously adjusting the pH of the solution and the concentration of OTG. Better separation can be achieved, however, when OTG is used, not as a sole chiral selector but rather together with sodium dodecyl sulfate (SDS) and cyclodextrin (CD). Interestingly, not only can this OTG-SDS-CD system provide better separation than OTG alone but also it can separate compounds which cannot be separated when only CD or CD and SDS are used. All amino acids were baseline separated with this OTG-SDS-CD system at optimal conditions.

Amino Acids↗
PubMed2002

Preparative-scale isoelectric trapping separations using a modified Gradiflow unit.

The Gradiflow BF200 preparative electrophoretic unit (Gradipore), which has been developed for size-based and charge-sign-based protein separations and in which the hydraulic flow path of the recirculating sample stream in the separation cartridge is orthogonal to the electric field, has been modified to carry out binary protein separations using the principles of isoelectric trapping. The disposable separation cartridge contained three isoelectric membranes which, along with the cartridge holder, formed the anode and cathode compartments and the anodic and cathodic separation compartments. The utility of the modified instrument was demonstrated by effecting a binary separation of chicken egg white across an isoelectric point 5.5 isoelectric membrane. The desalting and subsequent binary separation steps proved to be remarkably rapid, due to the favorable combination of short electrophoretic path, high electric field strength and large effective isoelectric membrane surface area.

Isoelectric Focusing↗
PubMed2003

Capillary-channeled polymer fibers as stationary phases in liquid chromatography separations.

A method utilizing capillary-channeled polymer (C-CP) fibers as stationary phases in high-performance liquid chromatographic separations has been investigated. Polymeric fibers of differing backbones (polypropylene and polyester) having nominal diameters of approximately 50 and approximately 35 microm and a channeled structure on their periphery were packed into stainless steel tubing (305 x 4.6 mm I.D.) for use in reversed-phase separations of various mixtures. The fibers have eight channels running continuously along the axis which exhibit very high surface activity. As such, solvent transport is affected through the channels through wicking action. Bundles of 1000-3000 fibers are loaded co-linearly into the tubing, providing flow channels extending the entire length of the columns. As a result, backing pressures are significantly lowered (approximately 50% reduction) in comparison to packed-sphere columns. In addition, the capital costs of the fiber material (< US$0.25 per column) are very attractive. Flow-rates of up to 5 ml/min can be used to achieve near baseline separation of related compounds in reasonable run times, indicating very fast mobile phase mass transfer (C-terms). The polymer stationary phases demonstrate high selectivity for a wide variety of analytes with gradient elution employed successfully in many instances. Specifically, separations of three polyaromatic hydrocarbons (benzo[a]pyrene, chrysene, pyrene), mixtures of both organic and inorganic lead compounds [chlorotriethyllead, chlorotriphenyllead, lead nitrate, lead(II) phthalocyanine], and a lipid standard of triglycerides were accomplished on the polymeric stationary phases. Other species of biological interest, including groups of aliphatic and aromatic amino acids have also been effectively separated. The reversed-phase nature of the fiber surfaces is supported through atomic force microscopy measurements using hydrophilic and hydrophobic functionalized polystyrene beads as the probe tips. Separations of the various analytes demonstrate the feasibility of utilizing C-CP fibers as stationary phases in reversed-phase LC. It is envisioned that columns of this nature would be particularly useful in prep-scale separations as well as for immobilization matrices for organic constituents in aqueous environments.

Chromatography, High Pressure Liquid↗
PubMed1997

Simple and rapid chromatographic method for the separation of major classes of ribosomal ribomononucleotides.

We have described a simple and rapid chromatographic method for the analytical and preparative separation of major types of ribosomal ribomononucleotides with Dowex 1-X10 (HCOO-, 37-74 microns) and Dowex 2-X10 (HCOO-, 37-74 microns) columns, by desorption with formiate solutions in 1-2 h. The separation has been achieved for Cp, Ap, Up and Gp, while a mixture of 2'-, and 3'-nucleoside phosphates desorbs as a single peak; with both resins, a successful separation was achieved with a load from 25 micrograms to 1 mg of ribomononucleotide mixture per ml of packed resin. A complete separation was achieved with Dowex 1, while the separation with Dowex 2 resin was even better. The resins cannot separate unusual nucleosides; therefore, our method is suitable for studies of ribonucleic acids with a low content of unusual nucleosides. Our method has been applied for the quantitative determination of the ribomononucleotide composition of 18S and 28S rRNAs, isolated from mammalian tissues: rat liver, mouse kidney and Ehrlich ascites cells. Dowex 1 and Dowex 2 resins afforded similar or identical ribomononucleotide compositions in all cases; analytical data were in agreement with the literature data. Our method is competitive, in several respects, with modern HPLC techniques for the separation of ribomononucleotides.

Animals↗
PubMed1997

Electroosmotic flow suppressing additives for capillary zone electrophoresis in a hydrodynamically closed separation system.

Electroosmotic flow in a hydrodynamically closed capillary zone electrophoresis (CZE) separation compartment must be minimized to achieve high efficiency CZE separations. A group of eight potential electroosmotic flow suppressors was investigated in this context for the separations in fluorinated ethylene-propylene capillary tubes. The suppressors included water soluble methylhydroxyethyl derivatives of cellulose, polyvinylalcohol, polyvinylpyrrolidones and polyethyleneglycols of different molecular masses and Triton X-100. Methylhydroxyethylcellulose derivatives and polyvinylalcohol were found to provide the highest separation efficiencies for a group of model anions when the electroosmotic flow suppressors were used as the carrier electrolyte additives. Using a methylhydroxyethylcellulose coated separation compartment very significant improvements in the separation efficiencies were achieved for polyvinylpyrrolidones and polyethyleneglycols applied in the carrier electrolyte solutions. For example, polyvinylpyrrolidone K 90 applied in this way gave for some of the model analytes the plate height values approaching those estimated in the calculations as theoretical limits for our experimental conditions (H approximately 3.5 microns). CZE experiments with albumin and gamma-globulin showed that the use of methylhydroxyethylcellulose derivative in the carrier electrolyte solution at pH = 9.2 was effective in eliminating potential disturbances in the separation efficiencies of the analytes due to adsorption of the proteins.

Anions↗
PubMed1999

Cyclodextrin-mediated micellar electrokinetic chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the enantiomer separation of racemorphan in human urine.

Micellar electrokinetic chromatography (MEKC) was successfully and conveniently applied to the chiral separation with the addition of cyclodextrins (CDs) as chiral selector to the running buffer. Chiral separation depended on the type of CD; in particular, beta-CD was effective for the chiral separation of racemorphan. We investigated the optimal conditions of type and concentration of CD as chiral selector for the routine enantiomeric separation of racemorphan with good reproducibility. The effects of other parameters such as buffer pH and detection wavelength were also investigated to obtain the optimum conditions for the enantiomeric separation of racemorphan. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used for confirmation of racemorphan. The optimal conditions for enantiomeric separation of the racemorphan were as follows: 50 mM borate buffer at pH 9.4 with 50 mM SDS, 10 mM beta-CD and 20% 1-propanol, 57 cm x 50 microns fused-silica capillary column, and UV detection at 192 nm. Based on the developed method, racemorphan in human urine was also separated and determined using solid-phase extraction and MEKC.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed2000

Retention mechanism, isocratic and gradient-elution separation and characterization of (co)polymers in normal-phase and reversed-phase high-performance liquid chromatography.

Synthetic (co)polymers or (co)oligomers with two (or more) repeating groups show not only molar mass distribution, but also composition and sequence distribution of the individual repeat units. To characterize such two- (or more-) dimensional distribution, liquid chromatography under "critical conditions" has been suggested, where the separation according to one type of repeating units is suppressed by balancing the adsorption and the size-exclusion effects. In present work it is shown that by combination of adequately selected separation conditions in normal-phase and in reversed-phase systems, the two-dimensional distribution mode can be adjusted to result in the separation following the distribution of any of the two repeat units in ethylene oxide-propylene oxide block (co)oligomers. Based on the retention mechanism suggested, prediction and optimization of the conditions for isocratic and gradient-elution separations of (co)oligomers is possible. HPLC-MS with atmospheric-pressure chemical ionization is a valuable tool for unambiguous identification of the individual (co)oligomers and their tracking in course of method development. Gradient elution can be used for the separation and characterization of block (co)oligomers of ethylene oxide (EO) and propylene oxide (PO) according to the number of the units in one block, while the separation according to the distribution of the units in the other block is suppressed. The effects of the arrangement of the individual EO and PO blocks in the block (co)oligomers (the sequence distribution) affects significantly the retention behavior and the selection of the optimum separation conditions.

Chromatography, High Pressure Liquid↗
PubMed2000

Separation and identification of etodolac and its urinary phase I metabolites using capillary electrochromatography and on-line capillary electrochromatography-electrospray ionisation mass spectrometry coupling.

Capillary high-performance liquid chromatography (capillary HPLC), pressure-assisted capillary electrochromatography (pCEC) and capillary electrochromatography (CEC) were performed in the same capillary packed with 5 microm octadecylsilica (C18) as stationary phase. These three separation modes were compared from the viewpoint of peak efficiency and separation selectivity in order to critically evaluate the advantages which CEC may offer compared to capillary HPLC for the solution of practical biomedical problems. The separation of the non-steroidal anti-inflammatory drug etodolac (ET, 1) and its phase I metabolites, 6-hydroxy etodolac (6-OH-ET, 2), 7-hydroxy etodolac (7-OH-ET, 3) and 8-(1'-hydroxyethyl) etodolac (8-OH-ET, 4) was selected as an example. Baseline separation of all compounds was achieved in different modes and conditions. The effect of pure electrophoretic separation mechanism on the overall separation selectivity observed in CEC has been shown. A high electroosmotic flow (EOF) was observed in C18 packed capillary even at pH 2.5 in various buffers. Furthermore, these separations were coupled on-line with electrospray ionisation mass spectrometry (ESI-MS) and the parent drug and its metabolites were identified in urine. For the coupling of CEC with ESI-MS a laboratory-made electrophoretic device was used in order to overcome some technical disadvantages of commercial instrumentation.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1998

Long-term effects of repeated maternal separation on three different latent inhibition paradigms.

In the present study we investigated the effect of repeated maternal separation on postnatal days 12, 14, 16, and 18 for 6 h/day on Wistar rats on three latent inhibition (LI) paradigms: two-way active avoidance, conditioned emotional response (CER), and conditioned taste aversion (CTA). In addition, hyperactivity induced by d-amphetamine and stereotypies induced by apomorphine were evaluated. In all three LI experiments, the control animals showed only marginal LI, whereas the maternally separated animals showed enhanced LI (only males in CTA). In two-way active avoidance within the nonpreexposed condition maternally separated animals showed improved acquisition of avoidance learning compared with the control animals. Sensitivity in response to amphetamine and apomorphine was not altered by the maternal separation procedure. Thus, maternal separation in this study, contrary to previous reports, but in line with results obtained following early handling before weaning, led to enhancement of the LI phenomenon as assessed in each of the three procedures. As our maternal separation procedure (6 h on days 12, 14, 16, and 18) led to behavioral outcomes that differed from those reported by Ellenbroek and Cools (24 h on day 10), it is suggested that maternal separation regimens that are dissimilar may lead to different and sometimes opposite behavioral effects.

Amphetamine↗
PubMed1997

Successful breast feeding in spite of early mother-baby separation for neonatal care.

OBJECTIVE: To study the effects on breast feeding duration of mother-baby separation after birth, owing to full-term babies' care in a neonatal unit for a maximum of six days. DESIGN: Retrospective review of medical records and telephone interviews with mothers. SETTING: A level II and III NICU in a University Hospital. PARTICIPANTS: 148 mother-baby pairs in a separated group and 3516 in a comparison group. MAIN OUTCOME MEASURES: Breast feeding success is defined as the duration of exclusive and partial breast feeding. FINDINGS: There were no differences in breast feeding duration between babies in the separated group and the comparison group. More than 70% of the babies in both groups were breast fed exclusively after two months and more than 50% for more than four months. In the separated group delays in physical contact, first suckling and regular breast feeding, and duration of separation were not associated with shorter duration of breast feeding. Mixed feeding, both breast and bottle at the time of discharge from hospital, and baby diagnosis of hypoglycaemia had a negative impact. Maternal level of education was also associated with the breast feeding outcome. CONCLUSIONS: Although every possible effort should be made to avoid mother-baby separation, women can be reassured that separation, because of their or the baby's medical condition during the first days after birth, need not be considered an obstacle to successful breast feeding.

Adolescent↗
PubMed2001

Cholesterol favors phase separation of sphingomyelin.

The phase behavior of mixed lipid dispersions representing the inner leaflet of the cell membrane has been characterized by X-ray diffraction. Aqueous dispersions of phosphatidylethanolamine:phosphatidylserine (4:1 mole/mole) have a heterogeneous structure comprising an inverted hexagonal phase H(II) and a lamellar phase. Both phases coexist in the temperature range 20-45 degrees C. The fluid-to-gel mid-transition temperature of the lamellar phase assigned to phosphatidylserine is decreased from 27 to 24 degrees C in the presence of calcium. Addition of sphingomyelin to phosphatidylethanolamine/phosphatidylserine prevents phase separation of the hexagonal H(II) phase of phosphatidylethanolamine but the ternary mixture phase separates into two lamellar phases of periodcity 6.2 and 5.6 nm, respectively. The 6.2-nm periodicity is assigned to the gel phase enriched in sphingomyelin of molecular species comprising predominantly long saturated hydrocarbon chains because it undergoes a gel-to-fluid phase transition above 40 degrees C. The coexisting fluid phase we assign to phosphatidylethanolamine and phosphatidylserine and low melting point molecular species of sphingomyelin which suppresses the tendency of phosphatidylethanolamine to phase-separate into hexagonal H(II) structure. There is evidence for considerable hysteresis in the separation of lamellar fluid and gel phases during cooling. The addition of cholesterol prevents phase separation of the gel phase of high melting point sphingomyelin in mixtures with phosphatidylserine and phosphatidylethanolamine. In the quaternary mixture the lamellar fluid phase, however, is phase separated into two lamellar phases of periodicities of 6.3 and 5.6 nm (20 degrees C), respectively. The lamellar phase of periodicity 5.6 nm is assigned to a phase enriched in aminoglycerophospholipids and the periodicity 6.3 nm to a liquid-ordered phase formed from cholesterol and high melting point molecular species of sphingomyelin characterized previously by ESR. Substituting 7-dehydrocholesterol for cholesterol did not result in evidence for lamellar phase separation in the mixture within the temperature range 20-40 degrees C. The specificity of cholesterol in creation of liquid-ordered lamellar phase is inferred.

Animals↗
PubMed1998

Chromatographic separations of aromatic carboxylic acids.

The purpose of this review is to present methods of chromatographic analysis of aromatic carboxylic acids. The separation, identification and quantitative analysis of aromatic carboxylic acids are necessary because of their importance as non-steroid antiphlogistic drugs, semi-products of biosynthesis of aromatic amino-acids in plants (phenolic acids), metabolites of numerous toxic substances, drugs and catecholamines. HPLC separation of ionic samples tends to be more complicated than separation of non-ionic compounds. The review describes the dependence of the retention of ionic solutes on pH and solvent composition as well as on the ionic strength of a mobile phase. The application of the ion-suppressing RP-HPLC method using organic modifiers (aqueous buffer solutions) as eluents in aromatic carboxylic acid analysis is also presented. In more difficult cases of analysis the addition of an ion-pairing reagent, such as the quaternary alkylammonium ion, is necessary to obtain satisfactory separations. Hypotheses of ion-pair formation in reversed-phase systems as well as the influence of various agents on the separation of ionic solutes in IP-RP systems are explained. Examples of the application of ion-pair liquid chromatography to the analysis of aromatic carboxylic acids have also been reviewed. The principles and application of ion-exchange chromatography to the purification, isolation and less frequently, to chromatographic analysis are discussed. Polar adsorbents and polar bonded stationary phases are also widely used in carboxylic acid separation in normal-phase systems, mainly by TLC, often coupled with densitometry. The review also shows examples of separation of chiral benzoic acids and their derivatives in LC systems. The possibilities of application of gas chromatography preceded by derivatisation or pyrolysis of acidic compounds and applications of GC-MS and Py-GC-MS coupled methods in identification and quantitation of aromatic carboxylic acids is also reviewed.

Carboxylic Acids↗
PubMed1998

Separation and quantitation of azimilide and its putative metabolites by capillary electrophoresis.

Reliable methods based on capillary electrophoresis (CE) have been developed for the separation and quantitation of azimilide, an antiarrhythmic drug under development at Procter & Gamble Pharmaceuticals (P&GP). Both capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) were employed in the separation of azimilide from its impurities, degradants and/or metabolites. Separation of azimilide from NE-11178, F-410, F-1054 and F-1292 was obtained by MECC at pH 9 with 50 mM sodium dodecyl sulfate (SDS). The separation of azimilide and NE-10171, a key metabolite of azimilide, was difficult because their structures differ by only a single methyl group. The best separation was achieved under acidic pH conditions with cetyltriethyl ammonium chloride (CTAC) additive in the buffer. All of the CE separations were completed within a substantially shorter time and with better resolution than the corresponding high-performance liquid chromatography (HPLC) separations. Quantitation was done with azimilide and NE-10171. Calibration curves ranging from 10 to 1000 microg/ml were obtained with R2 greater than 0.997 for both azimilide and NE-10171. The back-calculated concentrations of the calibration standards and the recoveries of the quality control (QC) samples were within the acceptance range currently used for HPLC methods. These results demonstrated the viability of CE as an alternative technique for drug metabolism studies in support of pharmaceutical development.

Anti-Arrhythmia Agents↗
PubMed2000

Optimal L(1)-L(2) primary tone level separation remains independent of test frequency in humans.

Previous studies described a systematic asymmetry of the level of the 2f(1)-f(2) distortion product otoacoustic emission (DP) in the space of the primary tones levels L(1) and L(2) in normal-hearing humans. Optimal primary tone level separations L(1)-L(2), which result in maximum DP levels, were close to L(1)=L(2) at high levels, but continuously increased with decreasing stimulus level towards L(1)>L(2) (Gaskill and Brown, 1990, J. Acoust. Soc. Am. 88, 821-839). At these optimal L(1)-L(2), however, not only DP levels in normal hearing were maximal, but also trauma-induced DP reductions. A linear equation that approximates optimal L(1)-L(2) level separations thus was suggested to be optimum for use in clinical applications (Whitehead et al., 1995, J. Acoust. Soc. Am. 97, 2359-2377). It was the aim of this study to extend the generality of optimal L(1)-L(2) separations to the typical human test frequency range for f(2) frequencies between 1 and 8 kHz. DPs were measured in 22 normal-hearing human ears at 61 primary tone level combinations, with L(2) between 5 and 65 dB SPL and L(1) between 30 and 70 dB SPL (f(2)/f(1)=1.2). It was found that the systematic dependence of the maximum DP level on the L(1)-L(2) separation is independent on frequency. Optimal L(1)-L(2) level separations may well be approximated by a linear equation L(1)=a L(2)+(1-a) b (after Whitehead et al., 1995) with parameters a=0.4 and b=70 dB SPL at f(2) frequencies between 1 and 8 kHz and L(2) levels between 20 and 65 dB SPL. Below L(2)=20 dB SPL, the optimal L(1) was found to be almost constant. Following previous notions (Gaskill and Brown, 1990), an analysis of basilar membrane response data in experimental animals (after Ruggero and Rich, 1991, Hear. Res. 51, 215-230) is further presented that relates optimal L(1)-L(2) separations to frequency-selective compression of the basilar membrane. Based on the assumption that optimal conditions for the DP generation are equal primary tone responses at the f(2) place, a linear increase of the optimal L(1)-L(2) level separation is graphically demonstrated, similar to our results in human ears.

Acoustic Stimulation↗
PubMed1997

Interactions between pulse separation and pulse polarity order in cochlear implants.

Interactions between pulse separation and pulse polarity order were examined using psychophysical studies of electrical detection thresholds in nonhuman primates. Subjects were trained using acoustic stimuli, then deafened in one ear and implanted with an electrode array for electrical stimulation of the cochlea. Threshold vs pulse separation functions for trains of biphasic electrical pulses were compared for constant and alternating leading phase polarity. When leading phase polarity was held constant, threshold vs pulse separation functions were nonmonotonic (U-shaped). Small polarity-dependent (cathodic vs anodic leading phase) differences in absolute thresholds were observed at long pulse separations, but function shape was independent of leading phase. When leading phase polarity alternated, there was a pronounced reduction in thresholds at short pulse separations (below about 1 ms), resulting in monotonically increasing threshold vs pulse separation functions. At long pulse separations, functions for alternating and constant polarity stimuli were similar. Polarity effects were most apparent for longer duration trains (20 pulses) at long pulse durations (1-2 ms/phase). For stimuli consisting of only two biphasic pulses, alternating polarity effects depended on whether cathodic or anodic phases were adjacent. The neural mechanisms underlying these effects probably include refractory properties and/or residual potentials.

Acoustic Stimulation↗
PubMed1999

Social separation in infant Cebus apella: patterns of behavioral and cortisol response.

34 infant tufted capuchins (Cebus apella) were separated from their social groups for a 2-hour period and videotaped in isolation at the ages of 6 months and 1 year. Baseline and 2-hour blood samples were measured for levels of serum cortisol. Compared to homecage baseline levels, passivity, locomotion and vocalizations increased during separation, while self-directed behavior and environmental exploration decreased. Both behavioral and cortisol responses to separation showed individual stability over the 6 month period, although both responses were somewhat attenuated at the later age. There was little correlation between cortisol and behavior during separations. Females vocalized more than did males during separations and showed greater cortisol increases at 6 months of age. The pattern of behavioral response seen in the 2 hours following separation appeared to be more passive than the typical 'protest' response described in many nonhuman primates, and may reflect either the physical circumstances of the separation or a characteristic of species with relaxed social bonds and considerable allomothering available to infants.

Animals↗
PubMed2001

The Arabidopsis myc/bHLH gene ALCATRAZ enables cell separation in fruit dehiscence.

BACKGROUND: Several processes of plant development, such as abscission, pollen release, fruit dehiscence, and seed dispersal, require organs or tissues to physically disassociate or split open. Due to the immobility of plant cells, these processes occur through coordinated mechanisms of cell separation that are not found in animals. Arabidopsis produces dry dehiscent fruits (siliques) making it a convenient system for the genetic study of cell separation associated with dehiscence. RESULTS: We describe here a novel mutation in Arabidopsis called alcatraz (alc), which prevents dehiscence of fruit by specifically blocking the separation of the valve cells from the replum. The ALC gene is shown to encode a protein related to the myc/bHLH family of transcription factors and is expressed in the valve margins of the silique, which is the site of cell separation during dehiscence. Detailed studies using TEM indicates that ALC enables cell separation in Arabidopsis fruit dehiscence by promoting the differentiation of a strip of labile nonlignified cells sandwiched between layers of lignified cells. Transgenic plants expressing antisense or dominant-negative ALC are defective in silique dehiscence. CONCLUSIONS: Cell separation in fruit dehiscence requires a specialized cell layer which is nonlignified and capable of autolysis, specified by a myc/bHLH protein encoded by ALC. These findings may have relevance to other processes requiring cell separation, as well as for the practical design of crops with reduced seed losses.

Amino Acid Sequence↗