The relative contributions of the three main intermediates of indole-3-acetic acid (IAA) biosynthesis from L-tryptophan (L-Trp); indole-3-pyruvate (IPyA), tryptamine (TNH2) and indole-3-acetaldoxime (IAOX), were investigated in vivo in tomato shoots. Initially, L-Trp, D-Trp, IPyA, TNH2 and IAA were purified from shoots, identified by full-scan mass spectrometry and their concentrations measured using gas chromatography with an electron capture detector. High specific activity [5-3H]IAOX and [5-3H]IPyA were synthesized from L-[5-3H]Trp and used as internal standards. Purification of endogenous IPyA was enabled by forming a stable pentafluorobenzyl oxime derivative in the crude plant extract. The respective endogenous concentrations of L-Trp, D-Trp, TNH2, IPyA and IAA were found to be 2,520, 103, 146.3, 5.9 and 8.5 ng g-1 f. wt. However, IAOX could not be identified as a natural constituent of tomato shoots by full-scan GC-MS. Secondly, incubation of tomato shoots for 6, 10 and 21 h in 30% 2H2O was used as a means of labelling IAA and its putative precursors in vivo. L-Trp, D-Trp, TNH2, IPyA and IAA were then extracted and purified and the 2H content measured by combined gas chromatography-mass spectrometry. These indole compounds were labelled rapidly with up to four 2H atoms. Direct comparison of the number and the amount of 2H atoms incorporated (pattern) was obtained from the mass spectral data on the common m/z 130 ion and its isotope peaks. IAA and L-Trp demonstrated an increase in 2H label with up to 17% and 21% of their molecules labelled at 10 h respectively. This was followed by a significant decrease in 2H label at 21 h to 12% for both L-Trp and IAA. This decrease in 2H label was attributed to an increase in protein catabolism, following shoot excision, resulting in the dilution of free L-Trp pool(s) with unlabelled L-Trp from which IAA is biosynthesized. This is reflected in the observed 1.6 to 1.8 fold increase of free L-Trp from 10 to 21 h. In contrast, tryptamine demonstrated a continual increase in 2H label with an average of 8, 20 and 28% of the molecules labelled at 6, 10 and 21 h respectively, suggesting that TNH2 and IAA were synthesized from separate Trp pools. In addition, the relatively slow rate at which 2H is incorporated into tryptamine would not be sufficient to account for the rate at which IAA becomes labelled. However, IPyA demonstrated a rapid increase in 2H with 22% and 37% of its molecules labelled at 6 and 10 h respectively. From the rate at which IPyA was labelled with 2H and the concentration of IPyA in tomato shoots a rate of synthesis for IPyA in tomato shoots was estimated which was sufficient to provide most of the shoot IAA requirements. Furthermore, the extent to which IAA and IPyA were labelled relative to that of total L-Trp would imply that a smaller more rapidly metabolised pool(s) of L-Trp was the precursor of these compounds. The rate and extent that D-Trp was labelled was consistently less than that of IAA precluding it as a possible precursor of IAA. These results indicate that in tomato shoots IAA is biosynthesized from a rapidly metabolized sub-pool(s) of L-trptophan predominantly via IPyA.

The relative contributions of the three main intermediates of indole-3-acetic acid (IAA) biosynthesis from L-tryptophan (L-Trp); indole-3-pyruvate (IPyA), tryptamine (TNH2) and indole-3-acetaldoxime (IAOX), were investigated in vivo in tomato shoots. Initially, L-Trp, D-Trp, IPyA, TNH2 and IAA were purified from shoots, identified by full-scan mass spectrometry and their concentrations measured using gas chromatography with an electron capture detector. High specific activity [5-3H]IAOX and [5-3H]IPyA were synthesized from L-[5-3H]Trp and used as internal standards. Purification of endogenous IPyA was enabled by forming a stable pentafluorobenzyl oxime derivative in the crude plant extract. The respective endogenous concentrations of L-Trp, D-Trp, TNH2, IPyA and IAA were found to be 2,520, 103, 146.3, 5.9 and 8.5 ng g-1 f. wt. However, IAOX could not be identified as a natural constituent of tomato shoots by full-scan GC-MS. Secondly, incubation of tomato shoots for 6, 10 and 21 h in 30% 2H2O was used as a means of labelling IAA and its putative precursors in vivo. L-Trp, D-Trp, TNH2, IPyA and IAA were then extracted and purified and the 2H content measured by combined gas chromatography-mass spectrometry. These indole compounds were labelled rapidly with up to four 2H atoms. Direct comparison of the number and the amount of 2H atoms incorporated (pattern) was obtained from the mass spectral data on the common m/z 130 ion and its isotope peaks. IAA and L-Trp demonstrated an increase in 2H label with up to 17% and 21% of their molecules labelled at 10 h respectively. This was followed by a significant decrease in 2H label at 21 h to 12% for both L-Trp and IAA. This decrease in 2H label was attributed to an increase in protein catabolism, following shoot excision, resulting in the dilution of free L-Trp pool(s) with unlabelled L-Trp from which IAA is biosynthesized. This is reflected in the observed 1.6 to 1.8 fold increase of free L-Trp from 10 to 21 h. In contrast, tryptamine demonstrated a continual increase in 2H label with an average of 8, 20 and 28% of the molecules labelled at 6, 10 and 21 h respectively, suggesting that TNH2 and IAA were synthesized from separate Trp pools. In addition, the relatively slow rate at which 2H is incorporated into tryptamine would not be sufficient to account for the rate at which IAA becomes labelled. However, IPyA demonstrated a rapid increase in 2H with 22% and 37% of its molecules labelled at 6 and 10 h respectively. From the rate at which IPyA was labelled with 2H and the concentration of IPyA in tomato shoots a rate of synthesis for IPyA in tomato shoots was estimated which was sufficient to provide most of the shoot IAA requirements. Furthermore, the extent to which IAA and IPyA were labelled relative to that of total L-Trp would imply that a smaller more rapidly metabolised pool(s) of L-Trp was the precursor of these compounds. The rate and extent that D-Trp was labelled was consistently less than that of IAA precluding it as a possible precursor of IAA. These results indicate that in tomato shoots IAA is biosynthesized from a rapidly metabolized sub-pool(s) of L-trptophan predominantly via IPyA.

The relative contributions of the three main intermediates of indole-3-acetic acid (IAA) biosynthesis from L-tryptophan (L-Trp); indole-3-pyruvate (IPyA), tryptamine (TNH2) and indole-3-acetaldoxime (IAOX), were investigated in vivo in tomato shoots. Initially, L-Trp, D-Trp, IPyA, TNH2 and IAA were purified from shoots, identified by full-scan mass spectrometry and their concentrations measured using gas chromatography with an electron capture detector. High specific activity [5-3H]IAOX and [5-3H]IPyA were synthesized from L-[5-3H]Trp and used as internal standards. Purification of endogenous IPyA was enabled by forming a stable pentafluorobenzyl oxime derivative in the crude plant extract. The respective endogenous concentrations of L-Trp, D-Trp, TNH2, IPyA and IAA were found to be 2,520, 103, 146.3, 5.9 and 8.5 ng g-1 f. wt. However, IAOX could not be identified as a natural constituent of tomato shoots by full-scan GC-MS. Secondly, incubation of tomato shoots for 6, 10 and 21 h in 30% 2H2O was used as a means of labelling IAA and its putative precursors in vivo. L-Trp, D-Trp, TNH2, IPyA and IAA were then extracted and purified and the 2H content measured by combined gas chromatography-mass spectrometry. These indole compounds were labelled rapidly with up to four 2H atoms. Direct comparison of the number and the amount of 2H atoms incorporated (pattern) was obtained from the mass spectral data on the common m/z 130 ion and its isotope peaks. IAA and L-Trp demonstrated an increase in 2H label with up to 17% and 21% of their molecules labelled at 10 h respectively. This was followed by a significant decrease in 2H label at 21 h to 12% for both L-Trp and IAA. This decrease in 2H label was attributed to an increase in protein catabolism, following shoot excision, resulting in the dilution of free L-Trp pool(s) with unlabelled L-Trp from which IAA is biosynthesized. This is reflected in the observed 1.6 to 1.8 fold increase of free L-Trp from 10 to 21 h. In contrast, tryptamine demonstrated a continual increase in 2H label with an average of 8, 20 and 28% of the molecules labelled at 6, 10 and 21 h respectively, suggesting that TNH2 and IAA were synthesized from separate Trp pools. In addition, the relatively slow rate at which 2H is incorporated into tryptamine would not be sufficient to account for the rate at which IAA becomes labelled. However, IPyA demonstrated a rapid increase in 2H with 22% and 37% of its molecules labelled at 6 and 10 h respectively. From the rate at which IPyA was labelled with 2H and the concentration of IPyA in tomato shoots a rate of synthesis for IPyA in tomato shoots was estimated which was sufficient to provide most of the shoot IAA requirements. Furthermore, the extent to which IAA and IPyA were labelled relative to that of total L-Trp would imply that a smaller more rapidly metabolised pool(s) of L-Trp was the precursor of these compounds. The rate and extent that D-Trp was labelled was consistently less than that of IAA precluding it as a possible precursor of IAA. These results indicate that in tomato shoots IAA is biosynthesized from a rapidly metabolized sub-pool(s) of L-trptophan predominantly via IPyA.

Malignant hyperthermia (MH) is a dominant skeletal muscle disorder caused by mutations in the ryanodine receptor skeletal muscle calcium release channel (RyR1). Allele-specific differences in RyR1 expression levels might provide insight into the observed incomplete penetrance and variations in MH phenotypes between individuals. Firstly, an H4833Y allele-specific PCR (AS-PCR) assay was designed that allowed for the relative quantification of the two RYR1 mRNA alleles in heterozygous samples. In four MHS skeletal muscle samples and two lymphoblastoid cell lines (LCLs), the wild type allele was found to be expressed at higher levels than the mutant RyR1 allele. These differences were not caused by variations in RYR1 mRNA stabilities. Secondly, high-throughput amplicon sequencing was employed for the quantification of both the T4826I and H4833Y causative MH mutations in heterozygous MHS samples. With the exception of one, all detected H4833Y and T4826I mutation frequencies were about 50%. This included a control, which was constructed and proven to have a 3:1 ratio of the wild type (H4833) versus the mutant (Y4833) RYR1 allele. This suggested that that the high-throughput amplicon sequencing approach as used here, was not suitable for accurate quantification of the two RyR1 alleles in heterozygous H4833Y MHS samples. To detect possible variations in RyR1 alleles at the protein level, the RyR1 was to be isolated from microsomes prepared from a H4833Y MHS frozen skeletal muscle tissue. Microsomes isolated from MHS skeletal muscle tissues lacked the immunoreactive band that was believed to be the full length RyR1. Poor muscle quality, due to long term storage was believed to be the main cause of RyR1 depletion. Faster and less expensive screening methodologies are required for the identification of genetic variants in MH research. Thus, in an additional project inexpensive and high-throughput high-resolution melting (HRM) assays were developed to allow screening of the RYR1 gene, for mutations associated with MH and/or central core disease (CCD).