Quantitative isotopic C nuclear magnetic resonance at natural abundance to probe enzyme reaction mechanisms via site-specific isotope fractionation: The case of the chain-shortening reaction for the bioconversion of ferulic acid to vanillin

Isotope fractionation is a powerful technique by which to probe the reaction mechanism of enzymes. The effect of a heavy isotope on the reaction energetics can be used to predict transition state architecture and reaction mechanism. In order to examine simultaneously the isotope fractionation in ¹³C at multiple sites within the substrate and product molecules without any need for site-selective isotope enrichment, a technique exploiting quantitative isotopic nuclear magnetic resonance (NMR) spectrometry at natural abundance (NAQ–NMR) has been developed. Here we report the first application of this technique to the study of an enzyme-catalyzed reaction, the bioconversion of ferulic acid to vanillin in cultures of Streptomyces setonii. We were able to show that the NAQ–NMR methodology is sufficiently precise and robust to measure the isotope shifts in the ¹³C/¹²C ratios in both substrate and product of this biotransformation, thereby permitting meaningful data to be obtained even at carbon positions that take part only indirectly in the reaction and show only secondary isotope fractionation. The results obtained provide direct evidence in support of the current hypothesis for the reaction mechanism of the enzyme hydroxycinnamoyl–CoA hydratase/lyase, notably the proposed involvement of the quinone methide enolate of feruloyl–CoA as intermediate in the catalytic pathway.     

The Multisubstrate Adapter Gab1 Regulates Hepatocyte Growth Factor (Scatter Factor)–c-Met Signaling for Cell Survival and DNA Repair

Hepatocyte growth factor (scatter factor) (HGF/SF) is a pleiotrophic mediator of epithelial cell motility, morphogenesis, angiogenesis, and tumorigenesis. HGF/SF protects cells against DNA damage by a pathway from its receptor c-Met to phosphatidylinositol 3-kinase (PI3K) to c-Akt, resulting in enhanced DNA repair and decreased apoptosis. We now show that protection against the DNA-damaging agent adriamycin (ADR; topoisomerase IIalpha inhibitor) requires the Grb2-binding site of c-Met, and overexpression of the Grb2-associated binder Gab1 (a multisubstrate adapter required for epithelial morphogenesis) inhibits the ability of HGF/SF to protect MDCK epithelial cells against ADR. In contrast to Gab1 and its homolog Gab2, overexpression of c-Cb1, another multisubstrate adapter that associates with c-Met, did not affect protection. Gab1 blocked the ability of HGF/SF to cause the sustained activation of c-Akt and c-Akt signaling (FKHR phosphorylation). The Gab1 inhibition of sustained c-Akt activation and of cell protection did not require the Gab1 pleckstrin homology or SHP2 phosphatase-binding domain but did require the PI3K-binding domain. HGF/SF protection of parental MDCK cells was blocked by wortmannin, expression of PTEN, and dominant negative mutants of p85 (regulatory subunit of PI3K), Akt, and Pak1; the protection of cells overexpressing Gab1 was restored by wild-type or activated mutants of p85, Akt, and Pak1. These findings suggest that the adapter Gab1 may redirect c-Met signaling through PI3K away from a c-Akt/Pak1 cell survival pathway.     

FXIII polymorphisms, fibrin clot structure and thrombotic risk

Fibrin clot structure is highly dependent on factor XIII activity. Activated FXIII catalyzes the formation of the peptide bonds between the gamma and alpha chains in noncovalently bound fibrin polymers and incorporates various adhesive and antifibrinolytic proteins into the final fibrin clot. In the absence of activated FXIII, clots are unstable and susceptible to fibrinolysis. Several studies have examined the effects of FXIII polymorphisms on final fibrin clot structure and clinical thrombotic risk. The Val34Leu FXIII polymorphism is associated with increased activation by thrombin. In the presence of saturating thrombin concentrations, however, FXIIIa specific enzyme activity is not affected by genetic polymorphisms. Fibrin clots formed in the presence of the FXIII 34Leu polymorphisms do tend to be thinner and less porous, however. The effects of prothrombin concentrations on clot structure have suggested that thinner clots are more resistant to fibrinolysis and associated with increased thrombotic risk. Most clinical studies of 34Leu FXIII carriers, however, have demonstrated a lower incidence of both venous and arterial thrombosis in carriers of the mutant allele compared to Val/Val carriers. One recent study has suggested that the interactions between FXIII phenotype and plasma fibrinogen concentrations significantly influence clinical thrombotic risk.     

RP2 and RPGR mutations and clinical correlations in patients with X-linked retinitis pigmentosa

We determined the mutation spectrum of the RP2 and RPGR genes in patients with X-linked retinitis pigmentosa (XLRP) and searched for correlations between categories of mutation and severity of disease. We screened 187 unrelated male patients for mutations, including 135 with a prior clinical diagnosis of XLRP, 11 with probable XLRP, 30 isolate cases suspected of having XLRP, and 11 with cone-rod degeneration. Mutation screening was performed by single-strand conformation analysis and by sequencing of all RP2 exons and RPGR exons 1-14, ORF15, and 15a. The refractive error, visual acuity, final dark-adapted threshold, visual field area, and 30-Hz cone electroretinogram (ERG) amplitude were measured in each patient. Among the 187 patients, we found 10 mutations in RP2, 2 of which are novel, and 80 mutations in RPGR, 41 of which are novel; 66% of the RPGR mutations were within ORF15. Among the 135 with a prior clinical diagnosis of XLRP, mutations in the RP2 and RPGR genes were found in 9 of 135 (6.7%) and 98 of 135 (72.6%), respectively, for a total of 79% of patients. Patients with RP2 mutations had, on average, lower visual acuity but similar visual field area, final dark-adapted threshold, and 30-Hz ERG amplitude compared with those with RPGR mutations. Among patients with RPGR mutations, those with ORF15 mutations had, on average, a significantly larger visual field area and a borderline larger ERG amplitude than did patients with RPGR mutations in exons 1-14. Among patients with ORF15 mutations, regression analyses showed that the final dark-adapted threshold became lower (i.e., closer to normal) and that the 30-Hz ERG amplitude increased as the length of the wild-type ORF15 amino acid sequence increased. Furthermore, as the length of the abnormal amino acid sequence following ORF15 frameshift mutations increased, the severity of disease increased.     

Synthesis of acetylcholine by lung cancer

The role of autocrine growth factors in the stimulation of lung cancer growth is well established. Nicotine is an agonist for acetylcholine receptors and stimulates lung cancer growth. This suggests that if lung cancers synthesize acetylcholine (ACh), then ACh may be an autocrine growth factor for lung cancer. Analysis of normal lung demonstrated that the cells of origin of lung cancers express the proteins necessary for non-neuronal ACh storage and synthesis. Analysis of mRNA from squamous cell lung carcinoma, small cell lung carcinoma (SCLC) and adenocarcinoma showed synthesis of choline acetyltransferase (ChAT) and nicotinic receptors. Immunohistochemical analysis of a retrospective series of SCLC and adenocarcinomas showed that more than 50% of the lung cancers screened expressed ChAT and nicotinic receptors. To study the effect of endogenous ACh synthesis on growth, SCLC cell lines were studied. SCLC cell lines were found to express ChAT mRNA and to secrete ACh into the medium as measured by HPLC separation and enzymatically-coupled electrochemical detection. The SCLC cell line NCI-H82 synthesized highest levels of ACh. Showing that the endogenously synthesized ACh interacted with its receptors to stimulate cell growth, addition of muscarinic and nicotinic antagonists slowed H82 cell proliferation. These findings demonstrate that lung cancer cell lines synthesize and secrete ACh to act as an autocrine growth factor. The existence of a cholinergic autocrine loop in lung cancer provides a basis for understanding the effects of nicotine in cigarette smoke on lung cancer growth and provides a new pathway to investigate for potential therapeutic approaches to lung cancer.     

Genetic modification removes an immunodominant allergen from soybean

The increasing use of soybean (Glycine max) products in processed foods poses a potential threat to soybean-sensitive food-allergic individuals. In vitro assays on soybean seed proteins with sera from soybean-sensitive individuals have immunoglobulin E reactivity to abundant storage proteins and a few less-abundant seed proteins. One of these low abundance proteins, Gly m Bd 30 K, also referred to as P34, is in fact a major (i.e. immunodominant) soybean allergen. Although a member of the papain protease superfamily, Gly m Bd 30 K has a glycine in the conserved catalytic cysteine position found in all other cysteine proteases. Transgene-induced gene silencing was used to prevent the accumulation of Gly m Bd 30 K protein in soybean seeds. The Gly m Bd 30 K-silenced plants and their seeds lacked any compositional, developmental, structural, or ultrastructural phenotypic differences when compared with control plants. Proteomic analysis of extracts from transgenic seed detected the suppression of Gly m Bd 30 K-related peptides but no other significant changes in polypeptide pattern. The lack of a collateral alteration of any other seed protein in the Gly m Bd 30 K-silenced seeds supports the presumption that the protein does not have a role in seed protein processing and maturation. These data provide evidence for substantial equivalence of composition of transgenic and non-transgenic seed eliminating one of the dominant allergens of soybean seeds.