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PubMedDate not supplied

Changes in content and composition of dietary fiber in yellow onions and red delicious apples during commercial storage.

Changes in pectin composition and solubility are part of the softening process in apples during ripening and postharvest storage. Lignification may also occur with long-term storage. In the United States, apples and onions are harvested once yearly and then stored and marketed for the next 12 months. The changes that occur in the dietary fiber content and composition in Red Delicious apples and yellow Spanish onions during storage were studied, and the loss of fiber in peeled apples was determined. Dietary fiber was extracted by the enzymatic-chemical method of Theander and Westerlund. Storage had no effect on total or insoluble fiber content of apples; Klason lignin concentration was greater in samples stored for 12 months than in those stored for 0, 4, or 8 months. Peeling reduced apple fiber concentration about 25% by decreasing neutral and acidic sugars and Klason lignin in the insoluble fraction. The total fiber content of onions increased with storage, primarily by increasing the insoluble fiber content of uronic acids. The results suggest that the standardized, environmentally controlled storage of apples, as used in Washington State, has little effect on dietary fiber content. In contrast, the less rigorously controlled storage conditions for yellow Spanish onions increases the insoluble fiber fraction and uronic acid content.

Arabinose↗
PubMed2004

Reduction of microbial pathogens during apple cider production using sodium hypochlorite, copper ion, and sonication.

Sodium hypochlorite (100 ppm), copper ion water (1 ppm), and sonication (22 to 44 kHz and 44 to 48 kHz) were assessed individually and in combination for their ability to reduce populations of Escherichia coli O157:H7 and Listeria monocytogenes on apples and in apple cider. Commercial unpasteurized cider was inoculated to contain approximately 10(6) CFU/ml of either pathogen and then sonicated at 44 to 48 kHz, with aliquots removed at intervals of 30 to 60 s for up to 5 min and plated to determine numbers of survivors. Subsequently, whole apples were inoculated by dipping to contain approximately 10(6) CFU/g E. coli O157:H7 or L. monocytogenes, held overnight, and then submerged in 1 ppm copper ion water with or without 100 ppm sodium hypochlorite for 3 min with or without sonication at 22 to 44 kHz and examined for survivors. Treated apples were also juiced, with the resulting cider sonicated for 3 min. Populations of both pathogens decreased 1 to 2 log CFU/ml in inoculated cider following 3 min of sonication. Copper ion water alone did not significantly reduce populations of either pathogen on inoculated apples. However, when used in combination with sodium hypochlorite, pathogen levels decreased approximately 2.3 log CFU/g on apples. Sonication of this copper ion-sodium hypochlorite solution at 22 to 44 kHz did not further improve pathogen reduction on apples. Numbers of either pathogen in the juice fraction were approximately 1.2 log CFU/ml lower after being juiced, with sonication (44 to 48 kHz) of the expressed juice decreasing L. monocytogenes and E. coli O157:H7 populations an additional 2 log. Hence, a 5-log reduction was achievable for both pathogens with the use of copper ion water in combination with sodium hypochlorite followed by juicing and sonication at 44 to 48 kHz.

Beverages↗
PubMed2006

Clinical effects of apple polyphenols on persistent allergic rhinitis: A randomized double-blind placebo-controlled parallel arm study.

BACKGROUND: We often encounter persistent allergic rhinitis due to house dust mites in the practice of otolaryngology, and its prevalence in Japan is high (18.7%). Persistent allergic rhinitis is usually treated with antihistamines and local steroids, but they often cause adverse effects such as sedation and drowsiness. Polyphenols derived from apples have been reported to suppress histamine release from rat cells, reduce auricular swelling in allergic mice, and alleviate skin inflammation in atopic patients. These effects suggest that apple polyphenols are effective for the treatment of various allergic disorders, but the results of their clinical use have not been reported. OBJECTIVE: To assess the effect of drinks containing apple polyphenols on clinical symptoms of persistent allergic rhinitis. METHODS: Thirty-three patients aged 15 to 65 years with moderate or severe persistent allergic rhinitis in whom the symptoms persisted for 3 years or longer were treated without apple polyphenols (control group), with a low dose of apple polyphenols, or with a high dose of apple polyphenols, and changes in the clinical symptoms were examined. RESULTS: Significant improvements were observed in sneezing attacks (P<.05) and nasal discharge (P<.01) in the high-dose group and in sneezing attacks (P<.05) in the low-dose group. Compared with the control group, an improvement was observed in sneezing attacks and nasal discharge in many patients of the polyphenol-treated groups. In terms of intranasal findings, a significant improvement was observed in swelling of the nasal turbinate in the low-dose group (P<.05). The percentage of patients who showed an improvement in swelling of the nasal turbinate was higher in the polyphenol-treated groups. CONCLUSIONS: We conclude that apple polyphenols are effective in alleviating symptoms of persistent allergic rhinitis.

Adult↗
PubMed1997

Outbreaks of Escherichia coli O157:H7 infection and cryptosporidiosis associated with drinking unpasteurized apple cider--Connecticut and New York, October 1996.

In October 1996, unpasteurized apple cider or juice was associated with three outbreaks of gastrointestinal illness. In the Western United States, an outbreak of Escherichia coli O157:H7 infections associated with unpasteurized commercial apple juice caused illness in 66 persons and one death. In addition, one outbreak of apple cider-related E. coli O157:H7 infections and another of cider-related Cryptosporidium parvum infections occurred in the Northeast. Apple cider is a traditional beverage produced and consumed in the fall. Cider often is manufactured locally at small cider mills where apples are crushed in presses, and the cider frequently is not pasteurized before sale. This report summarizes the clinical and epidemiologic features of the two apple cider-related outbreaks, which suggest that current practices for producing apple cider may not be adequate to prevent microbial contamination.

Adolescent↗
PubMed2005

Inhibitors of the epidermal growth factor receptor in apple juice extract.

The polyphenol-rich extract of a consumer-relevant apple juice blend was found to potently inhibit the growth of the human colon cancer cell line HT29 in vitro. The epidermal growth factor receptor (EGFR) and its subsequent signaling cascade play an important role in the regulation of cell proliferation in HT29 cells. The protein tyrosine kinase activity of an EGFR preparation was effectively inhibited by the polyphenol-rich apple juice extract. Treatment of intact cells with this extract resulted in the suppression of the subsequent mitogen-activated protein kinase cascade. Amongst the so far identified apple juice constituents, the proanthocyanidins B1 and B2 as well as quercetin-3-glc (isoquercitrin) and quercetin-3-gal (hyperoside) were found to possess substantial EGFR-inhibitory properties. However, as to be expected from the final concentration of these potential EGFR inhibitors in the original polyphenol-rich extract, a synthetic mixture of the apple juice constituents identified and available so far, including both proanthocyanidins and the quercetin glycosides, showed only marginal inhibitory effects on the EGFR. These results permit the assumption that yet unknown constituents contribute substantially to the potent EGFR-inhibitory properties of polyphenol-rich apple juice extract. In summary, the polyphenol composition of apple juice possesses promising growth-inhibitory properties, affecting proliferation-associated signaling cascades in colon tumor cells.

Beverages↗
PubMed2004

Cloning and functional expression of an ( E, E)-alpha-farnesene synthase cDNA from peel tissue of apple fruit.

Increased production of terpenes and many other aroma-related volatiles occurs with the onset of ripening in apple ( Malus domestica Borkh.) fruit. The gaseous plant hormone ethylene plays a key role in the induction of volatile synthesis, but the mechanism is not yet understood. Using a degenerate primer based on a short conserved sequence shared by several sesquiterpene synthases, reverse transcription-polymerase chain reaction with RNA isolated from peel tissue of 'Law Rome' apples yielded an approx. 800-bp gene fragment. This was used to screen a cDNA library generated from the peel tissue mRNA. A full-length terpene synthase (TS) cDNA 1,931 nucleotides long was isolated. The 1,728-bp open reading frame encodes a protein 576 amino acids long with a molecular mass of 66 kDa. Sequence analysis of the apple TS showed it to be most similar to several monoterpene synthases. Oddly, the TS includes an RR(X(8))W motif near the N-terminus that is common among monoterpene synthases but it lacks the plastid transit peptide sequence typically associated with genes of that group. Expression of the apple TS gene in Escherichia coli gave myc-epitope-tagged and untagged proteins estimated at approx. 68 and approx. 66 kDa, respectively. In assays of sesquiterpene synthase activity, with farnesyl diphosphate as substrate, the untagged bacterially expressed TS gene product synthesized ( E, E)-alpha-farnesene almost exclusively. In monoterpene synthase assays, with geranyl diphosphate as substrate, the untagged apple TS produced only ( E)-beta-ocimene, albeit at much reduced levels. Addition of a C-terminal myc tag appeared to completely prevent production of soluble protein under all of the expression conditions tested. This is the first report of an ( E, E)-alpha-farnesene synthase gene ( AFS1; GenBank accession number AY182241) from a flowering plant. RNA gel blots showed that AFS1 transcript increased about 4-fold in peel tissue of apple fruit during the first 4 weeks of storage at 0.5 degrees C. In contrast, when fruit were treated at harvest with 1-methylcyclopropene, a blocker of ethylene action, AFS1 mRNA declined sharply over the initial 4 weeks of cold storage, and fell to nearly undetectable levels by 8 weeks.

Alkyl and Aryl Transferases↗
PubMed2006

Radioprotective properties of apple polyphenols: an in vitro study.

Present study was undertaken to evaluate the radioprotective ability of total polyphenols extracted from edible portion (epicarp and mesocarp) of apple. Prior administration of apple polyphenols to murine thymocytes significantly countered radiation induced DNA damage (evaluated by alkaline halo assay) and cell death (trypan blue exclusion method) in a dose dependent manner maximally at a concentration of 2 and 0.2 mg/ml respectively. Apple polyphenols in a dose dependent fashion inhibited both radiation or Fenton reaction mediated 2-deoxyribose (2-DR) degradation indicating its ability to scavenge hydroxyl radicals and this activity was found to be unaltered in presence of simulated gastric juice. Similarly apple polyphenols in a dose dependent fashion scavenged DPPH radicals (maximum 69% at 1 mg/ml), superoxide anions (maximum 88% at 2 mg/ml), reduced Fe(3 +) to Fe(2 +) (maximum at 1 mg/ml) and inhibited Fenton reaction mediated lipid peroxidation (maximum 66% at 1.5 mg/ml) further establishing its antioxidative properties. Studies carried out with plasmid DNA revealed the ability of apple polyphenols to inhibit radiation induced single as well as double strand breaks. The results clearly indicate that apple polyphenols have significant potential to protect cellular system from radiation induced damage and ability to scavenge free radicals might be playing an important role in its radioprotective manifestation.

Animals↗
PubMed1991

Common epitopes of birch pollen and apples--studies by western and northern blot.

Eighty-three sera from patients with birch-pollen allergy were investigated for IgE antibodies against apple allergens by means of immunoblotting. In immunoblots, 81 patients (97.6%) exhibited IgE directed against the major allergen of birch, Bet v I (17 kd), and these patients also demonstrated IgE binding to apple allergens in the molecular weight range 17 to 18 kd. Inhibition studies by preincubation of sera with birch-pollen extract led to complete blocking of IgE binding to this 17 to 18 kd protein, whereas preincubation with apple extract could not diminish IgE binding to Bet V I. Furthermore, a 17 kd protein in apple extract could be detected by immunoblotting with a Bet v I-specific monoclonal antibody. Northern blotting with a Bet v I cDNA clone as a probe revealed cross-hybridization of birch and apple allergen coding nucleic acids under conditions of high stringency, suggesting significant homology of the nucleic acid level. Our results support the concept that antigens in birch pollen and apples share allergenic epitopes leading to IgE cross-reactivities that may cause clinical manifestations when a special threshold level of specific IgE antibodies is reached.

Adolescent↗
PubMed2007

Successful sublingual immunotherapy with birch pollen has limited effects on concomitant food allergy to apple and the immune response to the Bet v 1 homolog Mal d 1.

BACKGROUND: Cross-reactivity between the major birch pollen allergen, Bet v 1, and the apple protein, Mal d 1, frequently causes food allergy. OBJECTIVE: To investigate the effects of successful sublingual immunotherapy (SLIT) with birch pollen extract on apple allergy and the immune response to Bet v 1 and Mal d 1. METHODS: Before and after 1 year of SLIT, Bet v 1-sensitized patients with oral allergy syndrome to apple underwent nasal challenges with birch pollen and double-blind placebo-controlled food challenges with apple. Bet v 1-specific and Mal d 1-specific serum antibody levels and proliferation in PBMCs and allergen-specific T-cell lines (TCLs) were determined. Bet v 1-specific TCLs were mapped for T-cell epitopes. RESULTS: In 9 patients with improved nasal provocation scores to birch pollen, apple-induced oral allergy syndrome was not significantly reduced. Bet v 1-specific IgE and IgG(4) levels significantly increased. Bet v 1-specific T-cell responses to all epitopes and those cross-reactive with Mal d 1 significantly decreased. However, neither Mal d 1-specific IgE and IgG(4) levels nor Mal d 1-induced T-cell proliferation changed significantly. In contrast, Mal d 1-specific TCLs showed increased responses to Mal d 1 after 1 year of SLIT. CONCLUSION: This longitudinal study indicates that pollen SLIT does not efficiently alter the immune response to pollen-related food allergens, which may explain why pollen-associated food allergy is frequently not ameliorated by pollen immunotherapy even if respiratory symptoms significantly improve. CLINICAL IMPLICATIONS: SLIT with birch pollen may have no clinical effect on associated apple allergy.

Administration, Sublingual↗
PubMed2004

Multimodal assessment of cortical activation during apple peeling by NIRS and fMRI.

An intriguing application of neuroimaging is directly measuring actual human brain activities during daily living. To this end, we investigated cortical activation patterns during apple peeling. We first conducted a pilot study to assess the activation pattern of the whole lateral cortical surface during apple peeling by multichannel near-infrared spectroscopy (NIRS) and detected substantial activation in the prefrontal region in addition to expected activations extending over the motor, premotor and supplementary motor areas. We next examined cortical activation during mock apple peeling by simultaneous measurement using multichannel NIRS and functional magnetic resonance imaging (fMRI) in four subjects. We detected activations extending over the motor, premotor and supplementary motor areas, but not in the prefrontal cortex. Thus, we finally focused on the prefrontal cortex and examined its activation during apple peeling in 12 subjects using a multichannel NIRS. We subsequently found that regional concentrations of oxygenated hemoglobin significantly increased in the measured region, which encompassed portions of the dorsolateral, ventrolateral and frontopolar areas of the prefrontal cortex. The current study demonstrated that apple peeling as practiced in daily life recruited the prefrontal cortex but that such activation might not be detected for less laborious mock apple peeling that can be performed in an fMRI environment. We suggest the importance of cortical study of an everyday task as it is but not as a simplified form; we also suggest the validity of NIRS for this purpose. Studies on everyday tasks may serve as stepping stone toward understanding human activities in terms of cortical activations.

Activities of Daily Living↗
PubMed2006

A gene encoding starch branching enzyme I (SBEI) in apple (Malusxdomestica, Rosaceae) and its phylogenetic relationship to Sbe genes from other angiosperms.

An apple starch-branching enzyme SbeI gene (GenBank Accession No. DQ115404) has been isolated, cloned, and sequenced. The SbeI is a single copy gene in the apple genome, consisting of 14 exons and 13 introns, and covering 6075bp. As detected by RT-PCR, the apple SbeI is expressed at very low levels during early stages of fruit development; while, the highest levels of mRNA transcripts are observed at approximately 44 days post-pollination. Besides fruits, the apple SbeI is also expressed in buds and flowers, and very weakly in leaves. The genomic structure of SbeI in apple is strikingly similar to those reported so far in grasses (Poaceae), with exons 4 through 13 being of identical lengths in both apple and grasses. Moreover, structure similarities in exon lengths have also been detected in SbeII genes of both grasses and eudicots. These findings prompted the investigation of the evolutionary process of the Sbe gene family in angiosperms. A total of 26 Sbe sequences, representing an array of monocots and eudicots, are investigated in this study. Phylogenetic analysis has suggested that Sbe genes have duplicated into SbeI and SbeII prior to the divergence of moncots from eudicots. The SbeII gene is further duplicated into SbeIIa and SbeIIb prior to the radiation of grasses; however, it is not yet clear whether this duplication event has occurred before or after the radiation of the eudicots.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1998

Bet v 1, the major birch pollen allergen, and Mal d 1, the major apple allergen, cross-react at the level of allergen-specific T helper cells.

BACKGROUND: Food allergy to apple is frequent in individuals allergic to tree pollen. The major allergens of birch, Bet v 1, and apple, Mal d 1, have been cloned and sequenced and display a high degree of sequence identity, leading to IgE cross-reactivity. OBJECTIVE: We sought to investigate cross-reactivity between Bet v 1 and Mal d 1 at the level of allergen-specific T lymphocytes. METHODS: PBMCs of 13 patients allergic to birch pollen with oral allergy syndrome to apple were stimulated with rBet v 1 and rMal d 1, respectively, thereby establishing allergen-specific T-cell lines and T-cell clones. rMal d 1-specific T-cell cultures were tested for reactivity with rBet v 1, and rBet v 1-specific T cells were analyzed for reactivity with apple allergen. Cytokine production patterns in response to specific stimulation were evaluated. A selection of cross-reacting T-cell clones was mapped for epitope specificity by the use of overlapping Bet v 1- derived peptides. RESULTS: Nineteen Mal d 1-specific T-cell clones were produced, 79% of which cross-reacted with Bet v 1. Eight of 18 Bet v 1-specific T-cell clones cross-reacted with Mal d 1. Six peptides representing cross-reactive T-cell epitopes could be identified. The respective fragments from birch and apple displayed approximately 50% amino acid identity. Seventy percent of the cross-reactive T-cell clones revealed a T(H2)-like cytokine production pattern. CONCLUSION: The results indicate that cross-reactivity between apple and birch pollen leading to the clinical oral allergy syndrome occurs not only at the serologic, but also at the cellular level.

Adult↗
PubMed2002

Detection of four apple viruses by multiplex RT-PCR assays with coamplification of plant mRNA as internal control.

Two multiplex RT-PCR assays with specific coamplification of plant mRNA as an internal control from total nucleic acids are described for the parallel detection of Apple chlorotic leaf spot virus, Apple stem pitting virus, Apple mosaic virus and Apple stem grooving virus. All are important economically and common pathogens in commercial apple and pear cultivars, except for Apple mosaic virus. Four virus specific primer pairs and one primer pair which allows the specific amplification of mRNA of the mitochondrial nad5 gene are described. Specificity of all primer pairs was confirmed by sequencing the RT-PCR products. A range of different virus isolates from various geographic origins could be detected by these multiplex RT-PCR assays all year round. Viruses were detected reliably in composite extracts at a ratio of one part total nucleic acid extract from an infected sample mixed with 39 parts of extract from healthy samples. The use of the internal control minimizes the risk of obtaining false negative RT-PCR results, which is desirable for routine testing, and avoids the need to eliminate contaminating DNA in extracts. To our knowledge, this is the first report on the use of a specific internal RNA control from total nucleic acids. The multiplex RT-PCR assays described are reliable, rapid and sensitive methods for the detection of these viruses, and may replace techniques need commonly like indexing by woody indicators or ELISA.

Malus↗
PubMedDate not supplied

Light-stress-induced pigment changes and evidence for anthocyanin photoprotection in apples.

Fruit of two apple (Malus domestica Borkh.) cultivars, differing in their ability to produce anthocyanin pigments when exposed to sunlight, have been studied using reflectance spectroscopy. Comparison of the spectra shows that apple anthocyanins in vivo possess a symmetric absorption band at 500-600 nm with a maximum near 550 nm. Anthocyanins considerably increase light absorption by apples. In on-tree-ripening Zhigulevskoe apples, accumulating high amounts of anthocyanin pigments, chlorophyll contents in sunlit and shaded sides of the fruits are found to be similar. In contrast, frequently considerably lower chlorophyll content is estimated in sunlit compared with shaded sides of Antonovka apples exhibiting low potential for anthocyanin formation. Sunlight also brings about an increase of carotenoid content over that of chlorophylls and accumulation of substances responsible for light absorption in the range 350-400 nm. The rates of high-light-induced chlorophyll bleaching in red zones of fruit containing anthocyanins are considerably lower than those in green zones and decrease with an increase in the pigment content. Anthocyanins show more stability to irradiation than chlorophylls. A protective function of anthocyanins against both light-induced stress in, and damage to, apples is suggested. It is proposed that anthocyanins function as an effective internal light trap filling the chlorophyll absorption gap in the green-orange part of the visible spectrum.

Anthocyanins↗
PubMed2006

Allergenicity of 10 different apple varieties.

BACKGROUND: More than 100 apple varieties are consumed worldwide. However, the allergenic composition of most apple varieties has not been fully characterized. OBJECTIVE: To analyze the antigenic and allergenic profiles of 10 different commercially available apple varieties. METHODS: Golden, Golden Perlim, Reineta, Reineta Parda, Fuji, Verdedoncella, Granny-Smith, Pink Lady, Royal Gala, and Starking apple varieties were carefully peeled. Peels were individually extracted and centrifuged, and the supernatants were collected, dialyzed, filtered, frozen, and freeze-dried. The extracts were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblots. Protein content was measured using the Lowry-Biuret method. Twenty-two Spanish patients with oral allergy syndrome after apple ingestion were included in the study. Specific IgE to all apple varieties and birch pollen was evaluated. Eleven patients underwent skin prick testing using extracts of Golden, Starking, and Reineta Parda varieties to determine differences in their in vivo allergenicity. RESULTS: The antigenic profile of the 10 varieties showed differences using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A 9-kDa band, which could correspond to Mal d 3, was visualized in all the extracts. The Starking variety had the highest protein content. Higher specific IgE values were also obtained for the Starking variety, and the lowest values were for Reineta Parda and Royal Gala. The largest wheals were obtained in all patients with the Starking variety, and the smallest with Reineta Parda. Fourteen patients (64%) recognized Mal d 3 in the Golden variety using immunoblots; 6 patients (27%), who also had specific IgE to Betula, recognized bands with higher molecular weights. CONCLUSIONS: We demonstrated differences in the antigenic and allergenic profiles of the analyzed varieties and a significant variation in Mal d 3 content.

Adult↗
PubMed1996

Solid phase microextraction for quantitative headspace sampling of apple volatiles.

Solid phase microextraction (SPME) was evaluated for use in the quantification of aroma volatile production by Granny Smith apples during cool storage. Particular attention was paid to quantifying alpha-farnesene (3,7,11-trimethyldodeca-1,3(E),6(E),10-tetraene) due to its involvement in superficial scald, a disorder of cool stored apples. Comparison between SPME and solid phase extraction (SPE) showed that the SPME fiber had greater adsorption of high molecular weight (MW) volatiles such as alpha-farnesene. When sampling by SPME, these higher MW volatiles did not equilibrate between apples, headspace, and fiber within sampling times as long as 90 min, while lower MW volatiles equilibrated within 5 min. This behavior was also shown by a simple model system consisting of five selected volatiles dissolved in an involatile, lipophilic liquid (squalane). The less volatile high MW aroma compounds evaporated slowly from the surface of the apples and were depleted from the headspace because of very rapid adsorption by the SPME fiber. The amount of alpha-farnesene adsorbed by the fiber increased with air movement through the system. In a static headspace system, the amount of alpha-farnesene adsorbed by the fiber decreased nonlinearly with increasing distance from the apples, due to adsorption onto the glass walls. While SPME is ideal for rapid, qualitative determination of apple headspace volatiles, the slower equilibration of higher MW volatiles limits its use for quantification in more complex systems.

Chromatography, Gas↗
PubMed2000

Factor XI binding to activated platelets is mediated by residues R(250), K(255), F(260), and Q(263) within the apple 3 domain.

To localize the platelet binding site on factor XI, rationally designed, conformationally constrained synthetic peptides were used to compete with [(125)I]factor XI binding to activated platelets. The major platelet binding energy resided within the sequence of amino acids T(249)-F(260). Homology scanning, using prekallikrein amino acid substitutions within the synthetic peptide T(249)-F(260), identified a major role for R(250) in platelet binding. Inhibition of [(125)I]factor XI binding to activated platelets by the recombinant Apple 3 domain of factor XI and inhibition by unlabeled factor XI were identical, whereas the recombinant Apple 3 domain of prekallikrein had little effect. A "gain-of-function" chimera in which the C-terminal amino acid sequence of the Apple 3 domain of prekallikrein was replaced with that of factor XI was as effective as the recombinant Apple 3 domain of factor XI and unlabeled factor XI in inhibiting [(125)I]factor XI binding to activated platelets. Alanine scanning mutagenic analysis of the recombinant Apple 3 domain of factor XI indicated that amino acids R(250), K(255), F(260), and Q(263) (but not K(252) or K(253)) are important for platelet binding. Thus, the binding energy mediating the interaction of factor XI with platelets is contained within the C-terminal amino acid sequence of the Apple 3 domain (T(249)-V(271)) and is mediated in part by amino acid residues R(250), K(255), F(260), and Q(263).

Amino Acid Sequence↗
PubMed2000

Fast apple (Malus x domestica) and tobacco (Nicotiana tobacum) leaf polyphenol oxidase activity assay for screening transgenic plants.

A spectrophotometric assay method for the analysis of polyphenol oxidase (PPO), in apple and tobacco leaves, has been optimized to increase efficiency in the screening of large numbers of transgenic plants. Crude protein extracts from leaf punches were prepared in a FastPrep homogenizer. The addition of Triton X-100 during extraction resulted in 44 and 74% increases in the PPO activity recovered, from apple and tobacco, respectively. The enzyme kinetics differed markedly between apple and tobacco. Apple leaf PPO was isolated in a latent state and was activated by the addition of SDS. In contrast, tobacco PPO activity was inhibited by SDS, particularly at acidic pH. Apple PPO showed a pronounced pH optimum around pH 6, whereas the pH profile for tobacco PPO was much flatter, with a broad optimum around pH 4. The calculated Km' value for apple PPO, using 4-methylcatechol as substrate, was 8.1, and for tobacco the Km was 4.3. The PPO reaction was strongly inhibited by tropolone, a Cu competitor, and restored by the addition of Cu2+. Several factors affecting variability in leaf PPO activity levels in plants are discussed.

Biotechnology↗