Pharmacogenomics and nutrigenomics: synergies and differences.

The success of the Human Genome Project and the spectacular development of broad genomics tools have catalyzed a new era in both medicine and nutrition. The terms pharmacogenomics and nutrigenomics are relatively new. Both have grown out of their genetic forbears as large-scale genomics technologies have been developed in the last decade. The aim of both disciplines is to individualize or personalize medicine and food and nutrition, and ultimately health, by tailoring the drug or the food to the individual genotype. This review article provides an overview of synergies and differences between these two potentially powerful science areas. Individual genetic variation is the common factor on which both pharmacogenomics and nutrigenomics are based. Each human is genetically (including epigenetics) unique and phenotypically distinct. One of the expectations of both technologies is that a wide range of gene variants and related single-nucleotide polymorphism will be identified as to their importance in health status, validated and incorporated into genotype based strategies for the optimization of health and the prevention of disease. Pharmacogenomics requires rigorous genomic testing that will be regulated and analyzed by professionals and acted on by medical practitioners. As further information is obtained on the importance of the interaction of food and the human genotype in disease prevention and health, pharmacogenomics can provide an opportunity driver for nutrigenomics. As we move from disease treatment to disease prevention, the two disciplines will become more closely aligned.

Determination of the relative expression levels of rubisco small subunit genes in Arabidopsis by rapid amplification of cDNA ends.

Multigene families are common in higher organisms. However, due to the close similarities between members, it is often difficult to assess the individual contribution of each gene to the overall expression of the family. In Arabidopsis thaliana, there are four genes encoding the small subunits (SSU) of ribulose-1.5-bisphosphate carboxylase oxygenase (rubisco) whose nucleotide sequences are up to 98.4% identical. In order to overcome the technical limitations associated with gene-specific probes (or primers) commonly used in existing methods, we developed a new gene expression assay based on the RACE (rapid amplification of cDNA ends) technique with a single pair of primers. With this RACE gene expression assay, we were able to determine the relative transcript levels between four Arabidopsis SSU genes. We found that the relative SSU gene expression differed significantly between plants grown at different temperatures. Our observation raises the possibility that an adaptation of rubisco to the environment may be achieved through the specific synthesis of the SSU proteins, which is determined by the relative expression levels between the SSU genes.

A cysteine proteinase inhibitor purified from apple fruit.

A cysteine proteinase inhibitor has been purified from immature fruit of Malus domestica (var. Royal Gala). The M(r) of this apple cystatin is estimated to be 10,700 by MALDI-TOF mass spectrometry, 11 300 by SDS-PAGE and 11,000 by gel filtration. It is a relatively strong inhibitor of papain with a Ki value of 0.21 nM and also inhibits ficin and bromelain but not cathepsin B. An amino acid sequence was obtained from a peptide produced by trypsin digestion of the inhibitor. Comparison with other plant sequences shows a high degree of homology with other phytocystatins. As the single cysteine proteinase inhibitor detectable in immature apple fruit (5-8 mm diameter), levels of 83.3 pmol/g FW were determined. In larger fruit (up to 16 mm diameter) significantly less inhibitor was present (6.9 pmol/g FW). Given these low levels, it is postulated that this inhibitor has an endogenous role in apple fruit development rather than one of protection against pest or microbial attack.

Purification, characterization and cloning of an aspartic proteinase inhibitor from squash phloem exudate.

Phloem exudate from squash fruit contains heat-inactivated material which inhibits pepsin activity. This inhibitory activity was purified by mild acid treatment, chromatography on trypsin-agarose, Sephadex G-75 and reverse-phase HPLC, resulting in the elution of three peaks with pepsin-inhibitory activity. N-terminal sequencing indicated a common sequence of MGPGPAIGEVIG and the presence of minor species with seven- or two-amino-acid N-terminal extensions beyond this point. Microheterogeneity in this end sequence was exhibited within and between two preparations. Internal sequencing of a major peak after a trypsin digestion gave the sequence FYNVVVLEK. The common N-terminal sequence was used to design a degenerate primer for 3' rapid amplification of cDNA ends and cDNA clones encoding two isoforms of the inhibitor were obtained. The open reading frames of both cDNAs encoded proteins (96% identical) which contained the experimentally determined internal sequence. The amino acid content calculated from the predicted amino acid sequence was very similar to that measured by amino acid analysis of the purified inhibitor. The two predicted amino acid sequences (96 residues) had neither similarity to any other aspartic proteinase inhibitor nor similarity to any other protein. The inhibitors have a molecular mass of 10,552 Da, measured by matrix-assisted laser-desorption ionisation time-of-flight mass spectrometry and approximately 10,000 Da by SDS/PAGE, and behave as dimers of approximately 21,000 Da during chromatography on Superdex G-75 gel-filtration medium. The calculated molecular masses from the predicted amino acid sequences were 10,551 Da and 10,527 Da. The inhibitor was capable of inhibiting pepsin (Ki = 2 nM) and a secreted aspartic proteinase from the fungus Glomerella cingulata (Ki = 20 nM). The inhibitor, which is stable over acid and neutral pH, has been named squash aspartic proteinase inhibitor (SQAPI).

A Plant Chloroplast Glutamyl Proteinase.

A glutamyl proteinase was partially purified from Percoll gradient-purified spinach (Spinacia oleracea) chloroplast preparations and appeared to be predominantly localized in the chloroplast stroma. The enzyme degraded casein, but of the 11 synthetic endopeptidase substrates tested, only benzyloxycarbonyl-leucine-leucine-glutamic acid-[beta]-napthylamide was hydrolyzed at measurable rates. In addition, the enzyme cleaved the oxidized [beta]-chain of insulin after a glutamic acid residue. There was no evidence that native ribulose-1,5-bisphosphate carboxylase/oxygenase was cleaved by this proteinase. The apparent Km for benzyloxycarbonyl-leucine-leucine-glutamic acid-[beta]NA at the pH optimum of 8.0 was about 1 mM. Cl-ions were required for both activity and stability. Of the proteinase inhibitors covering all four classes of the endopeptidases, only 4-(2-aminoethyl)-benzenesulfonyl-fluoride HCl and L-1-chloro-3-[4-tosylamido]-4-phenyl-2-butanone significantly inhibited the proteinase. The partially purified enzyme had a molecular weight of about 350,000 to 380,000, based on size-exclusion chromatography. The enzyme has both similar and distinctive properties to those of the bacterial glutamyl proteinases. To our knowledge, this is the first description of a plant glutamyl proteinase found predominantly or exclusively in the chloroplast.

A method to distinguish between chemical shift and susceptibility effects in NMR microscopy and its application to insect larvae.

We propose a simple method of distinguishing Zeeman broadening arising from susceptibility inhomogeneity and chemical shift variation, applicable to NMR microscopy. The method is based on the use of a specially built probe-head in which orthogonal sample alignment is possible using the same radiofrequency (RF) coil. This allows the investigation of alignment effects in image distortion and relies on the fact that the isotropic chemical shift is invariant under reorientation, whereas the susceptibility-related local field will depend strongly on relative orientation of bounding surfaces with the external polarizing field. We apply this approach to the study of a simple phantom, and an insect larva (Spodoptera litura Fabricius), demonstrating in the latter case that susceptibility variations are sufficiently small to allow chemical shift imaging on a scale greater than 1 ppm.

Wounding induces a series of closely related trypsin/chymotrypsin inhibitory peptides in leaves of tobacco.

Wounding of tobacco (Nicotiana tabacum) leaves induced the expression of acid-stable trypsin/chymotrypsin inhibitory activity. Analysis by gel filtration determined that the inhibitory activity was contained within a fraction with a native M(r) of ca 5-7 x 10(3). Using ion-exchange column chromatography, this was resolved further into two major fractions, each of which inhibited both trypsin and chymotrypsin. Reverse-phase HPLC identified a total of six peptides from both fractions and each was purified to homogeneity. Four of these peptides inhibited both trypsin and chymotrypsin, a fifth inhibited trypsin only, while the sixth inhibited chymotrypsin almost exclusively. Sequencing of the N-terminal revealed that each peptide had an identical amino acid sequence and that these proteins are similar to a series of trypsin/chymotrypsin inhibitory peptides that are expressed predominantly in the stigmas of Nicotiana alata flowers.

Posttranslational modification of an isoinhibitor from the potato proteinase inhibitor II gene family in transgenic tobacco yields a peptide with homology to potato chymotrypsin inhibitor I.

A member of the potato proteinase inhibitor II (PPI-II) gene family under the control of the cauliflower mosaic virus 35S promoter has been introduced into tobacco (Nicotiana tabacum). Purification of the PPI-II protein that accumulates in transgenic tobacco has confirmed that the N-terminal signal sequence is removed and that the inhibitor accumulates as a protein of the expected size (21 kD). However, a smaller peptide of approximately 5.4 kD has also been identified as a foreign gene product in transgenic tobacco plants. This peptide is recognized by an anti-PPI-II antibody, inhibits the serine proteinase chymotrypsin, and is not observed in nontransgenic tobacco. Furthermore, amino acid sequencing demonstrates that the peptide is identical to a lower molecular weight chymotrypsin inhibitor found in potato tubers and designated as potato chymotrypsin inhibitor I (PCI-I). Together, these data confirm that, as postulated to occur in potato, PCI-I does arise from the full-length PPI-II protein by posttranslational processing. The use of transgenic tobacco represents an ideal system with which to determine the precise mechanism by which this protein modification occurs.

Photoinhibition of photosynthesis in intact kiwifruit (Actinidia deliciosa) leaves: Changes in susceptibility to photoinhibition and recovery during the growth season.

Kiwifruit (Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson) plants grown in an outdoor enclosure were exposed to the natural conditions of temperature and photon flux density (PFD) over the growing season (October to May). Temperatures ranged from 14 to 21° C while the mean monthly maximum PFD varied from 1000 to 1700 µmol · m(-2) · s(-1), although the peak PFDs exceeded 2100 µmol · m(-2) · s(-1). At intervals, the daily variation in chlorophyll fluorescence at 692 nm and 77K and the photon yield of O2 evolution in attached leaves was monitored. Similarly, the susceptibility of intact leaves to a standard photoinhibitory treatment of 20° C and a PFD of 2000 µmol · m(-2) · s(-1) and the ability to recover at 25° C and 20 µmol · m(-2) · s(-2) was followed through the season. On a few occasions, plants were transferred either to or from a shade enclosure to assess the suceptibility to natural photoinhibition and the capacity for recovery. There were minor though significant changes in early-morning fluorescence emission and photon yield throughout the growing season. The initial fluorescence, Fo, and the maximum fluorescence, Fm, were, however, significantly and persistently different from that in shade-grown kiwifruit leaves, indicative of chronic photoinhibition occurring in the sun leaves. In spring and autumn, kiwifruit leaves were photoinhibited through the day whereas in summer, when the PFDs were highest, no photoinhibition occurred. However, there was apparently no non-radiative energy dissipation occurring then also, indicating that the kiwifruit leaves appeared to fully utilize the available excitation energy. Nevertheless, the propensity for kiwifruit leaves to be susceptible to photoinhibition remained high throughout the season. The cause of a discrepancy between the severe photoinhibition under controlled conditions and the lack of photoinhibition under comparable, natural conditions remains uncertain. Recovery from photoinhibition, by contrast, varied over the season and was maximal in summer and declined markedly in autumn. Transfer of shade-grown plants to full sun had a catastrophic effect on the fluorescence characteristics of the leaf and photon yield. Within 3 d the variable fluorescence, Fv, and the photon yield were reduced by 80 and 40%, respectively, and this effect persisted for at least 20 d. The restoration of fluorescence characteristics on transfer of sun leaves to shade, however, was very slow and not complete within 15 d.

Photoinhibition of photosynthesis in intact kiwifruit (Actinidia deliciosa) leaves: effect of growth temperature on photoinhibition and recovery.

Intact leaves of kiwifruit (Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson) from plants grown in a range of controlled temperatures from 15/10 to 30/25°C were exposed to a photon flux density (PFD) of 1500 µmol·m(-2)·s(-1) at leaf temperatures between 10 and 25°C. Photoinhibition and recovery were followed at the same temperatures and at a PFD of 20 µmol·m(-2)·s(-1), by measuring chlorophyll fluorescence at 77 K and 692 nm, by measuring the photon yield of photosynthetic O2 evolution and light-saturated net photosynthetic CO2 uptake. The growth of plants at low temperatures resulted in chronic photoinhibition as evident from reduced fluorescence and photon yields. However, low-temperature-grown plants apparently had a higher capacity to dissipate excess excitation energy than leaves from plants grown at high temperatures. Induced photoinhibition, from exposure to a PFD above that during growth, was less severe in low-temperature-grown plants, particularly at high exposure temperatures. Net changes in the instantaneous fluorescence,F 0, indicated that little or no photoinhibition occurred when low-temperature-grown plants were exposed to high-light at high temperatures. In contrast, high-temperature-grown plants were highly susceptible to photoinhibitory damage at all exposure temperatures. These data indicate acclimation in photosynthesis and changes in the capacity to dissipate excess excitation energy occurred in kiwifruit leaves with changes in growth temperature. Both processes contributed to changes in susceptibility to photoinhibition at the different growth temperatures. However, growth temperature also affected the capacity for recovery, with leaves from plants grown at low temperatures having moderate rates of recovery at low temperatures compared with leaves from plants grown at high temperatures which had negligible recovery. This also contributed to the reduced susceptibility to photoinhibition in low-temperature-grown plants. However, extreme photoinhibition resulted in severe reductions in the efficiency and capacity for photosynthesis.

Photoinhibition of photosynthesis in intact kiwifruit (Actinidia deliciosa) leaves: Effect of temperature.

Photoinhibition of photosynthesis was induced in attached leaves of kiwifruit grown in natural light not exceeding a photon flux density (PFD) of 300 µmol·m(-2)·s(-1), by exposing them to a PFD of 1500 µmol·m(-2)·s(-1). The temperature was held constant, between 5 and 35° C, during the exposure to high light. The kinetics of photoinhibition were measured by chlorophyll fluorescence at 77K and the photon yield of photosynthetic O2 evolution. Photoinhibition occurred at all temperatures but was greatest at low temperatures. Photoinhibition followed pseudo first-order kinetics, as determined by the variable fluorescence (F v) and photon yield, with the long-term steady-state of photoinhibition strongly dependent on temperature wheareas the observed rate constant was only weakly temperature-dependent. Temperature had little effect on the decrease in the maximum fluorescence (F m) but the increase in the instantaneous fluorescence (F o) was significantly affected by low temperatures in particular. These changes in fluorescence indicate that kiwifruit leaves have some capacity to dissipate excessive excitation energy by increasing the rate constant for non-radiative (thermal) energy dissipation although temperature apparently had little effect on this. Direct photoinhibitory damage to the photosystem II reaction centres was evident by the increases in F o and extreme, irreversible damage occurred at the lower temperatures. This indicates that kiwifruit leaves were most susceptible to photoinhibition at low temperatures because direct damage to the reaction centres was greatest at these temperatures. The results also imply that mechanisms to dissipate excess energy were inadequate to afford any protection from photoinhibition over a wide temperature range in these shade-grown leaves.

Photoinhibition of photosynthesis in intact kiwifruit (Actinidia deliciosa) leaves: Recovery and its dependence on temperature.

Recovery of photoinhibition in intact leaves of shade-grown kiwifruit was followed at temperatures between 10° and 35° C. Photoinhibition was initially induced by exposing the leaves for 240 min to a photon flux density (PFD) of 1 500 µmol·m(-2)·s(-1) at 20° C. In additional experiments to determine the effect of extent of photoinhibition on recovery, this period of exposure was varied between 90 and 400 min. The kinetics of recovery were followed by chlorophyll fluorescence at 77K. Recovery was rapid at temperatures of 25-35° and slow or negligible below 20° C. The results reinforce those from earlier studies that indicate chilling-sensitive species are particularly susceptible to photoinhibition at low temperatures because of the low rates of recovery. At all temperatures above 15° C, recovery followed pseudo first-order kinetics. The extent of photoinhibition affected the rate constant for recovery which declined in a linear fashion at all temperatures with increased photoinhibition. However, the extent of photoinhibition had little effect on the temperature-dependency of recovery. An analysis of the fluorescence characteristics indicated that a reduction in non-radiative energy dissipation and repair of damaged reaction centres contributed about equally to the apparent recovery though biochemical studies are needed to confirm this. From an interpretation of the kinetics of photoinhibition, we suggest that recovery occurring during photoinhibition is limited by factors different from those that affect post-photoinhibition recovery.

Photoinhibition of photosynthesis in intact kiwifruit (Actinidia deliciosa) leaves: Effect of light during growth on photoinhibition and recovery.

Photoinhibition of photosynthesis was induced in intact kiwifruit (Actinidia deliciosa (A. Chev.) C. F. Liang et A. R. Ferguson) leaves grown at two photon flux densities (PFDs) of 700 and 1300 µmol·m(-2)·s(-1) in a controlled environment, by exposing the leaves to PFD between 1000 and 2000 µmol·m(-2)·s(-1) at temperatures between 10 and 25°C; recovery from photoinhibition was followed at the same range of temperatures and at a PFD between 0 and 500 µmol·m(-2)·s(-1). In either case the time-courses of photoinhibition and recovery were followed by measuring chlorophyll fluorescence at 692 nm and 77K and by measuring the photon yield of photosynthetic O2 evolution. The initial rate of photoinhibition was lower in the high-light-grown plants but the long-term extent of photoinhibition was not different from that in low-light-grown plants. The rate constants for recovery after photoinhibition for the plants grown at 700 and 1300 µmol·m(-2)·s(-1) or for those grown in shade were similar, indicating that differences between sun and shade leaves in their susceptibility to photoinhibition could not be accounted for by differences in capacity for recovery during photoinhibition. Recovery following photoinhibition was increasingly suppressed by an increasing PFD above 20 µmol·m(-2)·s(-1), indicating that recovery in photoinhibitory conditions would, in any case, be very slow. Differences in photosynthetic capacity and in the capacity for dissipation of non-radiative energy seemed more likely to contribute to differences in susceptibility to photoinhibition between sun and shade leaves of kiwifruit.

Ribulose 1,5-bisphosphate carboxylase. Effect on the catalytic properties of changing methionine-330 to leucine in the Rhodospirillum rubrum enzyme.

Oligonucleotide-directed mutagenesis of cloned Rhodospirillum rubrum ribulose bisphosphate carboxylase/oxygenase with a synthetic 13mer oligonucleotide primer was used to effect a change at Met-330 to Leu-330. The resultant enzyme was kinetically examined in some detail and the following changes were found. The Km(CO2) increased from 0.16 to 2.35 mM, the Km(ribulose bisphosphate) increased from 0.05 to 1.40 mM for the carboxylase reaction and by a similar amount for the oxygenase reaction. The Ki(O2) increased from 0.17 to 6.00 mM, but the ratio of carboxylase activity to oxygenase activity was scarcely affected by the change in amino acid. The binding of the transition state analogue 2-carboxyribitol 1,5-bisphosphate was reversible in the mutant and essentially irreversible in the wild type enzyme. Inhibition by fructose bisphosphate, competitive with ribulose bisphosphate, was slightly increased in the mutant enzyme. These data suggest that the change of the residue from methionine to leucine decreases the stability of the enediol reaction intermediate.

Activity expressed from cloned Anacystis nidulans large and small subunit ribulose bisphosphate carboxylase genes.

The ribulose bisphosphate carboxylase/oxygenase (EC4.1.1.39) (RubisCO) large and small subunit genes from Anacystis nidulans have been cloned as a single fragment into M 13mp10 and pEMBL8 and expressed in Escherichia coli. From M 13mp10 a low yield of enzyme with high specific activity was obtained. The molecular weight of the active enzyme was 260 000 Da and of the inactive enzyme approximately 730 000 Da. The small and large subunits cloned separately did not express activity. The RubisCO gene cloned into pEMBL8 expressed activity up to 22 times that from the M 13 cloned RubisCO DNA. The RubisCO protein produced by the pEMBL cloned gene had a normal MW (550 000). Immunoprecipitation and polyacrylamide gel electrophoresis showed the presence of both large and small subunits.

Effects of manganese ions and magnesium ions on the activity of soya-bean ribulose bisphosphate carboxylase/oxygenase.

The Michaelis constants of soya-bean ribulose bisphosphate carboxylase for CO2 in the carboxylation reaction and for O2 in the oxygenation reaction depend on the nature of the bivalent cation present. In the presence of Mg2+ the Km for bicarbonate is 2.48 mM, and the Km for O2 is 37% (gas-phase concentration). With Mn2+ the values decrease to 0.85 mM and 1.7% respectively. For the carboxylation reaction Vmax. was 1.7 mumol/min per mg of protein with Mg2+ but only 0.29 mumol/min per mg of protein with Mn2+. For the oxygenation reaction, Vmax. values were 0.61 and 0.29 mumol/min per mg of protein respectively with Mg2+ and Mn2+.

Carbon Dioxide Fixation by Lupin Root Nodules: II. Studies with C-labeled Glucose, the Pathway of Glucose Catabolism, and the Effects of Some Treatments That Inhibit Nitrogen Fixation.

Labeling studies using detached lupin (Lupinus angustifolius) nodules showed that over times of less than 3 minutes, label from [3,4-(14)C]glucose was incorporated into amino acids, predominantly aspartic acid, to a much greater extent than into organic acids. Only a slight preferential incorporation was observed with [1-(14)C]- and [6-(14)C]glucose, while with [U-(14)C]-glucose more label was incorporated into organic acids than into amino acids at all labeling times. These results are consistent with a scheme whereby the "carbon skeletons" for amino acid synthesis are provided by the phosphoenolpyruvate carboxylase reaction.A comparison of (14)CO(2) release from nodules supplied with [1-(14)C]- and [6-(14)C]glucose indicated that the oxidative pentose phosphate pathway accounted for less than 6% of glucose metabolism. Several enzymes of the oxidative pentose phosphate and glycolytic pathways were assayed in vitro using the 12,000g supernatant fraction from nodule homogenates. In all cases, the specific activities were adequate to account for the calculated in vivo fluxes.Three out of four diverse treatments that inhibited nodule nitrogen fixation also inhibited nodule CO(2) fixation, and in the case of the fourth treatment, replacement of N(2) with He, it was shown that the normal entry of label from exogenous (14)CO(2) into the nodule amino acid pool was strongly inhibited.

A kinetic study of ribulose bisphosphate carboxylase from the photosynthetic bacterium Rhodospirillum rubrum.

The activation kinetics of purified Rhodospirillum rubrum ribulose bisphosphate carboxylase were analysed. The equilibrium constant for activation by CO(2) was 600 micron and that for activation by Mg2+ was 90 micron, and the second-order activation constant for the reaction of CO(2) with inactive enzyme (k+1) was 0.25 X 10(-3)min-1 . micron-1. The latter value was considerably lower than the k+1 for higher-plant enzyme (7 X 10(-3)-10 X 10(-3)min-1 . micron-1). 6-Phosphogluconate had little effect on the active enzyme, and increased the extent of activation of inactive enzyme. Ribulose bisphosphate also increased the extent of activation and did not inhibit the rate of activation. This effect might have been mediated through a reaction product, 2-phosphoglycolic acid, which also stimulated the extent of activation of the enzyme. The active enzyme had a Km (CO2) of 300 micron-CO2, a Km (ribulose bisphosphate) of 11--18 micron-ribulose bisphosphate and a Vmax. of up to 3 mumol/min per mg of protein. These data are discussed in relation to the proposed model for activation and catalysis of ribulose bisphosphate carboxylase.