Plant proteases for bioactive peptides release: A review.

Proteins are a potential source of health-promoting biomolecules with medical, nutraceutical, and food applications. Nowadays, bioactive peptides production, its isolation, characterization, and strategies for its delivery to target sites are a matter of intensive research. In vitro and in vivo studies regarding the bioactivity of peptides has generated strong evidence of their health benefits. Dairy proteins are considered the richest source of bioactive peptides, however proteins from animal and vegetable origin also have been shown to be important sources. Enzymatic hydrolysis has been the process most commonly used for bioactive peptide production. Most commercial enzymatic preparations frequently used are from animal (e.g., trypsin and pepsin) and microbial (e.g., Alcalase® and Neutrase®) sources. Although the use of plant proteases is still relatively limited to papain and bromelain from papaya and pineapple, respectively, the application of new plant proteases is increasing. This review presents the latest knowledge in the use and diversity of plant proteases for bioactive peptides release from food proteins including both available commercial plant proteases as well as new potential plant sources. Furthermore, the properties of peptides released by plant proteases and health benefits associated in the control of disorders such as hypertension, diabetes, obesity, and cancer are reviewed.

SU-E-T-128: Dosimetric Characteristics of Gafchromic EBT3 Films for Megavoltage Photon and Proton Beams.

PURPOSE: Gafchromic film for quantitative analysis was renewed from EBT2 to EBT3 film in November 2011. The purpose of this study is to investigate the relevant characteristics of EBT3 film for its application in dosimetric verification for IMRT/VMAT or proton therapy. METHOD: We investigated the characteristics of EBT3 film with comparison of previous EBT2 film. The experiments in this study composed two categories. At first, the photo spectroscopy for the irradiated film was compared between EBT2 and EBT3. The film 1 day after the irradiation was analyzed by a photo spectrometer (SR520: JASCO Corporation, Japan). Secondly, we investigated several calibration curves which obtained by same batch. The films were calibrated by irradiation the films to 13 dose steps. The irradiated films were scanned by a flatbed scanner (ES-10000XL, Epson-Seiko Corporation, Japan). The difference on scan orientation was evaluated alternate portrait and landscape directions. The photon and proton beams were delivered from Clinac 21EX (Varian) and Mitsubishi machine, respectively. RESULTS: The peak absorption wavelength of EBT3 film and its response at all active range were basically same with that of EBT2 film. The peak wavelength of photo absorption in EBT3 was observed at 585 and 634 nm. The fog optical density was increased due to the hazy matte polyester for active layer. However, there is no change the tendency of the calibration curve responding to megavoltage photon and proton beams. The scan orientation dependency of EBT3 film was observed with similar to EBT2 film. The optical density of portrait orientation was 10% higher than that of landscape orientation. CONCLUSION: The dosimetric characteristics of EBT3 film were basically same with EBT2 film. With regard to the matte polyester, the creation of Newton's rings during scanning procedure was reduced. However, the suitable scan protocol should be used for accurate film dosimetry.

SU-E-T-196: Commissioning for Volumetric Modulated Radiation Therapy on Varian Clinac 21EX.

PURPOSE: Recently the volumetric arc therapy (VMAT) technology such as RapidArc is widely distributed in Japan. These technologies are normally provided by the high spec linear accelerator such as Trilogy, Novalis Tx, Synergy, et al. The specific DICOM-file is generally used for commissioning of these technologies. On the other hand, we had to apply RapidArc into historic linear accelerator. This title expresses an experience how we performed the commissioning of RapidArc with the old linear accelerator. METHODS: Two Varian's linear accelerators "Clinac 21EX" equipped with Millenium multi-leaf collimator and a Varian's treatment planning system "Eclipse ver.8.9" were used for this study. The commissioning for RapidArc was performed in energy 4,6,10,15 MV (Max-DR: 250, 600, 400, 600 MU/min). Commissioning procedure composed two categories: the general machine QA for DMLC-IMRT procedure and the specific RapidArc QA procedure. In RapidArc QA procedure, we modified DICOM-file to apply into the potential spec of Clinac 21EX optimally. The specific MLC-motion sequence and the gantry rotation speed were created by the dedicated programs (Shaper and DicomEdit, Varian) for RapidArc QA procedure. Each tolerance value was defied by the data from daily/monthly QA and the paper by Ling et al. RESULTS: As the results of the general machine QA procedure, the variance of radiation output during static/dynamic gantry rotation was less than 1%. The deference of fence tests during static/dynamic gantry rotation and RapidArc were less than 1 mm in each. However, the results of the RapidArc QA were worse than the latest machine (especially variable gantry speed) and it was careful to define tolerance level. CONCLUSION: The procedure of commissioning for RapidArc on historic linear accelerator was proposed. Several minor revisions for DICOM-file should be required for suitable commissioning and it may ensure the tolerance limit for gantry/MLC-leaf motion speeds.

Expression profiles of genes associated with viral entry in HCV-infected human liver.

Recent studies have demonstrated that several cellular factors are involved in entry of hepatitis C virus (HCV) into host cells. Detailed gene expression profiles of these factors in HCV-infected livers have not been reported for humans. Transcriptional levels of LDL receptor (LDLR), CD81, scavenger receptor class B type I (SR-BI), claudin-1, and occludin genes in liver samples from patients with chronic hepatitis C were investigated. Serum levels of LDL-cholesterol (LDL-C) and HCV core antigen were also evaluated, and expression of claudin-1 and occludin were immunohistochemically analyzed. Compared with normal liver, transcription of LDLR and claudin-1 genes was significantly suppressed (P < 0.0001) and occludin transcription was significantly up-regulated in HCV-infected livers (P < 0.0001). Significant positive correlations were found for LDLR versus occludin, LDLR versus claudin-1, occludin versus claudin-1, and CD81 versus SR-BI in HCV-infected (P = 0.0012, P < 0.0001, P = 0.0004, and P < 0.0001, respectively) and normal livers (P < 0.0001, P = 0.0051, P < 0.0001, and P < 0.0001, respectively). Positive correlation was observed between serum levels of HCV core antigen and LDL-C (P = 0.0147), with their levels negatively correlated to LDLR (P = 0.0270 and P = 0.0021, respectively). Immunohistochemically, hepatocellular expression of claudin-1 and occludin was increased in HCV-infected livers. Different levels of expression were demonstrated at the mRNA and protein levels for occludin and claudin-1 in HCV-infected and normal livers. Correlation of elements associated with viral entry was comparable in HCV-infected and normal livers.

Influence of an electric field on oriented films of DMPC/gramicidin bilayers: a circular dichroism study.

A film of oriented bilayers containing a mixture of gramicidin and dimyristoylphosphatidylcholine (DMPC) has been deposited on a fused-silica window coated with a 10 nm thick gold layer. The thin layer of gold allows the application of an electric potential across the film and the study of its influence on the structure and integrity of the bilayers. Electrochemical measurements, ellipsometry, and far-UV circular dichroism (CD) were employed to characterize the properties of the film of bilayers as a function of the potential applied to the gold electrode. For potentials across the film that are within the range approximately +300 to -150 mV the oriented film of bilayers is stable, and no change in the CD spectra of gramicidin molecule is observed. At more negative potentials, an increase in the film thickness and water content measured by ellipsometry indicated that the film swells and incorporates water, which causes a change in the circular dichroism spectrum of gramicidin molecules in the film. This transformation was interpreted as a change in the average orientation of gramicidin molecules within the film due to a decrease in the ordering of the molecules upon swelling.

Therapeutic effect of bezafibrate against biliary damage: a study of phospholipid secretion via the PPARalpha-MDR3 pathway.

OBJECTIVE: Bezafibrate (BF) has been used to treat biliary damage, particularly in patients with primary biliary cirrhosis (PBC), and its clinical efficacy has been demonstrated. The mechanism of action is thought to involve activation of the PPARalpha-MDR3-phospholipid (PL) secretion pathway. We tried to confirm this hypothesis in patients with hepatobiliary disease. METHODS: The levels of serum gamma-glutamyl transpeptidase and alkaline phosphatase, and those of bile components were examined before and after BF administration in patients with obstructive jaundice undergoing percutaneous transhepatic biliary drainage (PTBD). Hepatic expression of PPARalpha and MDR3 was quantified by real-time PCR in patients with PBC or non-alcoholic fatty liver disease (NAFLD). RESULTS: In patients with obstructive jaundice, BF decreased the serum levels of biliary enzymes and increased the bile concentration of PL. In patients with PBC or NAFLD, the expression levels of MDR3 were already up-regulated before starting the BF treatment. Although BF treatment did not further up-regulate MDR3 expression in NAFLD patients, PPARalpha expression was significantly increased. CONCLUSIONS: BF enhanced the secretion of PL into bile in cholestatic patients undergoing PTBD. However, in patients with PBC or NAFLD, diseases that represent cholesterol overload, MDR3 was already expressed at a high level to compensate for bile acids overproduction, and its expression was hardly affected by BF. In patients with chronic liver diseases such as PBC, BF may induce clinical effects via mechanisms independent of PL secretion.

N-terminal portion acts as an initiator of the inactivation of pepsin at neutral pH.

Porcine pepsin, an aspartic protease, is unstable at neutral pHs where it rapidly loses activity, however, its zymogen, pepsinogen, is stable at neutral pHs. The difference between the two is the presence of the prosegment in pepsinogen. In this study, possible factors responsible for instability were investigated and included: (i) the distribution of positively charged residues on the surface, (ii) an insertion of a peptide in the C-terminal domain and (iii) the dissociation of the N-terminal fragment of pepsin. Mutations to change the number and the distribution of positive charges on the surface had a minor effect on stability. No effect on stability was observed for the deletion of a peptide from the C-terminal domain. However, mutations on the N-terminal fragment had a major impact on stability. At pH 7.0, the N-fragment mutant was inactivated 5.8 times slower than the wild-type. The introduction of a disulfide bond between the N-terminal fragment and the enzyme body prevented the enzyme from denaturing. The above results showed that the inactivation of pepsin was initiated by the dissociation of the N-fragment and that the sequence of this portion was a major determinant for enzyme stability. Through this study, we have created porcine pepsin with increased pH stability at neutral pHs.

The pepsin residue glycine-76 contributes to active-site loop flexibility and participates in catalysis.

Glycine residues are known to contribute to conformational flexibility of polypeptide chains, and have been found to contribute to flexibility of some loops associated with enzymic catalysis. A comparison of porcine pepsin in zymogen, mature and inhibited forms revealed that a loop (a flap), consisting of residues 71--80, located near the active site changed its position upon substrate binding. The loop residue, glycine-76, has been implicated in the catalytic process and thought to participate in a hydrogen-bond network aligning the substrate. This study investigated the role of glycine-76 using site-directed mutagenesis. Three mutants, G76A, G76V and G76S, were constructed to increase conformational restriction of a polypeptide chain. In addition, the serine mutant introduced a hydrogen-bonding potential at position 76 similar to that observed in human renin. All the mutants, regardless of amino acid size and polarity, had lower catalytic efficiency and activated more slowly than the wild-type enzyme. The slower activation process was associated directly with altered proteolytic activity. Consequently, it was proposed that a proteolytic cleavage represents a limiting step of the activation process. Lower catalytic efficiency of the mutants was explained as a decrease in the flap flexibility and, therefore, a different pattern of hydrogen bonds responsible for substrate alignment and flap conformation. The results demonstrated that flap flexibility is essential for efficient catalytic and activation processes.

Effect of processing conditions on phospholipase D activity of corn kernel subcellular fractions.

The influence of physicochemical conditions on the phospholipase D (PLD) activity of subcellular preparations of sweet corn (Zea mays L. cv. Peaches and Cream) kernels has been studied. The microsomal, mitochondrial, and cytosolic preparations of corn kernels possessed PLD activity albeit at varying proportions. The microsomal and cytosolic PLD activities were stimulated 2-fold between 5 and 15 degrees C. Ethanol had varying modulatory effects on PLD activity. By contrast, acetaldehyde was a potent inhibitor of PLD. As well, a naturally occurring C(6) aldehyde such as hexanal and an alcohol such as hexanol inhibited PLD activity efficiently. Divalent cations such as calcium chloride and magnesium chloride stimulated PLD activity at micromolar levels. Monovalent cations such as KCl and NaCl did not appear to affect PLD activity. Partial purification of PLD from the microsomal, mitochondrial, and cytosolic fractions separately revealed four major isoforms with relative molecular masses of 200, 140-150, 102-108, and 60-66 kDa. The importance of PLD in the maintenance of processed food quality is discussed.

Contribution of a prosegment lysine residue to the function and structure of porcine pepsinogen A and its active form pepsin A.

A conserved lysine residue, Lys36p, on the prosegment of pepsinogen was replaced with a positively charged arginine (K36pR), a negatively charged glutamic acid (K36pE), and a neutral side chain methionine (K36pM). K36pM and K36pE mutants were extremely unstable and degraded rapidly, especially K36pE, which was inactivated during purification. This instability was confirmed by microcalorimetry where the denaturing temperatures for K36pM and K36pE were 6 degrees C and 10 degrees C lower than the wild-type, respectively. As a function of pH, K36pM and K36pR were activated over a broader range of pH as compared with wild-type. The mutant pepsinogens were activated faster than wild-type with K36pM being activated approximately 10 times faster. The activated pepsins from the various mutant pepsinogens showed lower kinetic efficiency than wild-type enzyme. Catalytic rate constants were reduced by half. The results suggested Lys36p is important for the correct folding of the active-centre residues. The molecular modeling calculation suggested that the position of Asp215 was substantially altered. In conclusion, the above results would suggest that Lys36p was important not only for stability of the prosegment and pepsinogen, but also for the correct alignment of the active-centre residues.

The role of the flap residue, threonine 77, in the activation and catalytic activity of pepsin A.

Flexible loops, often referred to as flaps, have been shown to play a role in catalytic mechanisms of different enzymes. Flaps at the active site regions have been observed in the crystal structures of aspartic proteinases and their residues implicated in the catalytic processes. This research investigated the role of the flap residue, threonine 77, in the activation of pepsinogen and the catalytic mechanism of pepsin. Three mutants, T77S, T77V and T77G, were constructed. Differences in amino acid polarity and hydrogen bonding potential were shown to have an influence on the activation and catalytic processes. T77S activated at the same rate and had similar catalytic parameters as the wild-type pepsin. The activation rates of T77V and T77G were slower and their catalytic efficiencies lower than the wild-type. The results demonstrated that the threonine 77 polar side chain played a role in a proteolysis. The contribution of the side chain to zymogen activation was associated with the proteolytic cleavage of the prosegment. It was postulated that the hydroxyl group at position 77 provided an essential hydrogen bond that contributed to proper substrate alignment and, indirectly, to a catalytically favorable geometry of the transition state.

Mechanism of activation of the gastric aspartic proteinases: pepsinogen, progastricsin and prochymosin.

The gastric aspartic proteinases (pepsin A, pepsin B, gastricsin and chymosin) are synthesized in the gastric mucosa as inactive precursors, known as zymogens. The gastric zymogens each contain a prosegment (i.e. additional residues at the N-terminus of the active enzyme) that serves to stabilize the inactive form and prevent entry of the substrate to the active site. Upon ingestion of food, each of the zymogens is released into the gastric lumen and undergoes conversion into active enzyme in the acidic gastric juice. This activation reaction is initiated by the disruption of electrostatic interactions between the prosegment and the active enzyme moiety at acidic pH values. The conversion of the zymogen into its active form is a complex process, involving a series of conformational changes and bond cleavage steps that lead to the unveiling of the active site and ultimately the removal and dissociation of the prosegment from the active centre of the enzyme. During this activation reaction, both the prosegment and the active enzyme undergo changes in conformation, and the proteolytic cleavage of the prosegment can occur in one or more steps by either an intra- or inter-molecular reaction. This variability in the mechanism of proteolysis appears to be attributable in part to the structure of the prosegment. Because of the differences in the activation mechanisms among the four types of gastric zymogens and between species of the same zymogen type, no single model of activation can be proposed. The mechanism of activation of the gastric aspartic proteinases and the contribution of the prosegment to this mechanism are discussed, along with future directions for research.

Engineered porcine pepsinogen exhibits dominant unimolecular activation.

An engineered pepsinogen, which was a fusion protein of thioredoxin and pepsinogen, exhibited dominant self-activation (unimolecular reaction; intramolecular activation) in contrast to recombinant pepsinogen which exhibited both unimolecular and bimolecular reactions (intermolecular activation mediated by pepsin released during activation). At pH values of 1.1, 2.0, and 3.0, activation curves for the engineered pepsinogen were hyperbolic rather than sigmoidal, indicating that self-activation was the dominant activation mechanism in comparison to the slower bimolecular activation. To confirm which activation mechanism was dominant, an equal mole of pepsin was added to accelerate the bimolecular reaction during activation. The addition of exogenous pepsin did not affect the activation rate of the engineered pepsinogen but accelerated pepsinogen activation through the bimolecular reaction. The above results indicated that the engineered pepsinogen exhibited, primarily, a self-activation mechanism and that bimolecular activation was negligible.

Kinetic model for carbon partitioning in Solanum tuberosum tubers stored at 2 degrees C and the mechanism for low temperature stress-induced accumulation of reducing sugars.

Exposure to low but nonfreezing temperatures induces the breakdown of starch and the accumulation of sucrose, glucose and fructose in potato tubers, a complex phenomenon known as low-temperature sweetening (LTS). A kinetic model for the degradation of starch to sucrose, fructose, glucose, hexose phosphates and carbon dioxide in 2 degrees C-stored mature Solanum tuberosum cv. Norchip (LTS-sensitive) and Solanum tuberosum seedlling ND860-2 (LTS-tolerant) tubers is presented in this work. Analysis of sugar accumulation data in tubers grown in 1993 and 1994 showed no significant differences in the rates of conversion of starch to hexose phosphates and hexose phosphates to sucrose for both cultivars (P > 0.05). The rate constant corresponding to invertase activity was 2.3 day(-1) for Norchip tubers and 1.1 day(-1) for ND860-2 tubers grown in 1993 (P < or = 0.05); however, no significant differences were observed in invertase activity for 1994-grown tubers (P > 0.05). The accumulation of the reducing sugars fructose and glucose was found to be dependent on the relative difference in rate constants corresponding to invertase activity and glycolytic/respiratory capacity. This difference was 3-4 fold greater for Norchip in 1993, and 4-6 fold greater for Norchip in 1994, than for ND860-2 (P < or = 0.05). Results from the analysis also suggest that the amount of available starch for degradation was greater in Norchip tubers than ND860-2 tubers (P < or = 0.05). Our analysis suggests that tubers with decreased invertase activity coupled to increased glycolytic/respiratory capacity should be more tolerant to low-temperature stress.

Some physicochemical and functional properties of cowpea (Vigna unguiculata) isoelectric protein isolate as a function of pH and salt concentration.

Cowpea isoelectric protein isolate was prepared by alkali extraction followed by acid precipitation at pH 4.5. Physicochemical and functional properties of the isolate were determined at various combinations of pH (3-8) and NaCl concentrations (0.5-2.0 M). Results showed that at all NaCl concentrations, emulsifying activity index, heat coagulability, aromatic hydrophobicity (ARH) and fluorescence intensity (FI) were high at low pH and gradually decreased with increasing pH. Protein solubility and foam stability were low at low pH and high NaCl concentrations, but increased with increase in pH. The relationships of ARH, FI and UV-absorption maxima (Amax) with the functional properties were determined using multiple regression analyses. Significant (P < or = 0.05) equations describing the functional properties on the basis of ARH, FI and Amax were generated; however, the contribution of these parameters was low as indicated by the low correlation coefficients. Regression results suggest that other physicochemical properties (e.g. electrostatic interactions) were also important contributors to the functional properties examined.

Improving the thermostability of Bacillus stearothermophilus neutral protease by introducing proline into the active site helix.

A proline residue was introduced into the N-terminus (Ile140 and Asp141), the middle (Leu147) and the C-terminus (Asp153) of the active site helix of Bacillus stearothermophilus neutral protease for comparing the effects on the thermostability. Introduction of a proline residue into the N-terminus at sites 140 and 141 increased the half-survival temperature (HST) by 7.5 and 2.8 degrees C, respectively, from 68.3 degrees C of the wild-type enzyme. A proline residue at Leu147 decreased the HST by 10.2 degrees C, while no change was observed by introducing a proline residue in the C-terminus. These results were coincidental with the CD data which indicated increases in Tm values of 4.4 and 2.3 degrees C for I140P and D141P, respectively. Susceptibility to alpha-chymotrypsin hydrolysis markedly decreased in mutants I140P and D141P, while increasing in L147P. Molecular modeling suggested that glycine residues on the N-terminus side of proline residues in I140P and D141P relaxed the possible strain caused by proline introduction. The thermostability can, therefore, be explained based on changes in the molecular rigidity.

Low-temperature stress induces transient oscillations in sucrose metabolism in Solanum tuberosum.

Exposure to low but nonfreezing temperatures induces the net breakdown of starch and the accumulation of sucrose, glucose and fructose in potato tuber tissue, a complex phenomenon known as low-temperature sweetening (LTS). When transferred to 4 degrees C storage, tissue sucrose levels in LTS-sensitive potato tubers (Solanum tuberosum cv. Norchip) did not change monotonically to a new steady state, but rather transiently oscillated about the trajectory to the new steady state. The dynamic patterns observed in sensitive tubers grown in 1993 and 1994 were qualitatively similar. Quantitatively, however, the transient oscillation had a period of 11.5 days in 1993, whereas a period of 80 days was observed in 1994. In contrast, the sucrose levels of the LTS-tolerant potato tubers (Solanum tuberosum seedling ND860-2) increased monotonically to a higher level upon exposure to low temperatures.

Expression of soluble cloned porcine pepsinogen A in Escherichia coli.

A system for the production of soluble porcine pepsinogen A (EC 3.4.23.1) was developed by fusing the pepsinogen and thioredoxin genes and then expressing the fused product (Trx-PG) in Escherichia coli. The expressed fusion protein was purified using a combination of ion-exchange and hydrophobic chromatography. Trypsin digestion of the fusion protein yielded pepsinogen which was one residue longer than the intrinsic length. Acidification of either the fusion protein or pepsinogen (tryptic digestion of Trx-PG) yielded recombinant pepsin A (r-pepsin). When compared with commercial porcine pepsin A, r-pepsin had similar milk-clotting and proteolytic activities, kinetic parameters and pH dependency. The above results indicate that an expression system was developed which yielded fully active soluble pepsin(ogen) from Escherichia coli.

The sole lysine residue in porcine pepsin works as a key residue for catalysis and conformational flexibility.

Pepsin contains a single lysine residue which protrudes from the enzyme's surface, behind the active site cleft, on the C-terminal domain. Mutations of pepsin by site-directed mutagenesis of the Lys-319 residue were generated to study the structure-function relationships. Kinetic parameters, pH activity profiles, along with conformational analysis using circular dichroism (CD), and molecular modelling were examined for the wild-type (non-mutant) and mutant enzymes. The pepsin mutations, Lys-319-->Met and Lys-319-->Glu, resulted in a progressive increase in the Km and similar decrease in kcat, respectively, as well as being denatured at a lower pH than the wild-type pepsin. CD analysis indicated that mutations at Lys-319 resulted in changes in secondary structure fractions which were reflected in changes in enzymatic activity as compared to the wild-type pepsin, i.e. kinetic data and pH denaturation study. Molecular modelling of mutant enzymes indicated differences in flexibility in the flap loop region of the active site, the region around the entrance of the active site cleft, sub-site regions for peptide binding, and in the subdomains of the C-terminal domain when compared to the wild-type enzyme. The results suggest that Lys-319, which is distal to the active site, is important to the flexibility/stability of the enzyme, as well as to its catalytic machinery.

Potential for improvement by selection for reducing sugar content after cold storage for three potato populations.

The objectives of this study were to examine the expected response to selection for reducing-sugar content after cold storage in three hybrid populations, to determine whether these populations included clones low in reducing sugars, and to investigate the effectiveness of indirect selection for chip colour based on selection of sugar content after cold storage. The three hybrid populations included: a random sample of 39 clones of Population 1, which was derived from crossing ND860-2 (a clone low in reducing sugars) with F58089 (a clone intermediate in reducing sugars); 40 clones of Population 2, which was obtained from crossing ND860-2 with Russette (a clone high in reducing sugars); and 40 clones of Population 3, which was derived from crossing Russette with F58089. Sugar content and chip colour were assessed in tubers stored for 2 months at 4 °C at Cambridge, Ontario, and at 3 °C at Benton Ridge, New Brunswick. Population 1 had a slightly greater predicted response to selection for reduction in glucose and total reducing sugars than the other two populations. This could be attributed to higher heritability estimates for Population 1, which was a reflection of smaller clone × environment interaction mean squares. The greater potential advance by selection for fructose, glucose, and total reducing sugars, was a direct consequence of its lower means for these traits. Low reducing-sugar clones were found in all three populations, indicating their potential use for the selection of low reducing sugars. Populations 2 and 3, however, would require stronger selection pressures and, therefore, large population sizes. Expected correlated responses for chip colour by selection for fructose and glucose were similar to, and sometimes exceeded, the expected direct responses in all three populations. Indirect responses for chip colour by selection for sucrose, however, were lower than direct selection responses. These results indicate that indirect selection for chip colour, by selection for either fructose or glucose content after cold storage, is as effective as direct selection for chip colour.