Enzyme models: design and selection

There are two approaches to the discovery of enzyme mimics, that is identifying molecules that are able to bind substrate(s) and then catalyze reactions. The first approach, often inspired by enzymes themselves, utilises chemical knowledge and experience to design the catalyst. The other approach is to create a library and select the best host of a transition state analogue of the required reaction.     

Sterol C-24 methyltransferase type 1 controls the flux of carbon into sterol biosynthesis in tobacco seed

The first committed step in the conversion of cycloartenol into Delta(5) C24-alkyl sterols in plants is catalyzed by an S-adenosyl-methionine-dependent sterol-C24-methyltransferase type 1 (SMT1). We report the consequences of overexpressing SMT1 in tobacco (Nicotiana tabacum), under control of either the constitutive carnation etched ring virus promoter or the seed-specific Brassica napus acyl-carrier protein promoter, on sterol biosynthesis in seed tissue. Overexpression of SMT1 with either promoter increased the amount of total sterols in seed tissue by up to 44%. The sterol composition was also perturbed with levels of sitosterol increased by up to 50% and levels of isofucosterol and campesterol increased by up to 80%, whereas levels of cycloartenol and cholesterol were decreased by up to 53% and 34%, respectively. Concomitant with the enhanced SMT1 activity was an increase in endogenous 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, from which one can speculate that reduced levels of cycloartenol feed back to up-regulate 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and thereby control the carbon flux into sterol biosynthesis. This potential regulatory role of SMT1 in seed sterol biosynthesis is discussed.     

Prevalence of risk factors for cardiovascular disease in Canadians 55 to 74 years of age: results from the Canadian Heart Health Surveys, 1986-1992

BACKGROUND: By 2016, the proportion of Canadians older than 65 years of age will increase to 16%, and there will be an increase in the absolute number of cases of cardiovascular disease in older Canadians. The Canadian Heart Health Surveys database provides information about this population upon which health policy related to cardiovascular disease can be based. This paper presents for the first time population-based data on the risk factors for cardiovascular disease in older Canadians. METHODS: Canadians from all 10 provinces participated in surveys of cardiovascular risk factors; health insurance registries were used as sampling frames. In each province, probability samples of 2200 adults 18 to 74 years old not living in institutions, on reserves or in military camps were asked to participate in interviews and to undergo testing at clinics for major risk factors for cardiovascular disease. RESULTS: A total of 2739 men (response rate 70%) and 2617 women (response rate 66%) aged 55 to 74 years participated in the survey and also provided follow-up clinical measurements at the clinic. Overall, 52% of participants were hypertensive, 26% had isolated systolic hypertension, and 30% had a total blood cholesterol level of 6.2 mmol/L or greater. Rates of current smoking were lower in women than men (17% v. 22%). Overall, 87% of men and 78% of women who were current smokers smoked at least 10 cigarettes per day. Only slightly more than half of participants exercised at least once a week for at least 15 minutes, and almost half had a body mass index of 27 or greater. In only 4% was no major risk factor for cardiovascular disease detected. INTERPRETATION: Significant numbers of older Canadians have one or more major risk factors for cardiovascular disease. Many of these risk factors are amenable to modification.     

Conversion of lipids to fatty alcohols and lysolipids by NaBH4

A variety of fatty acid esters were reacted with NaBH₄ in tetra-hydrofuran (THF) — water mixtures. Although triglycerides and free acids were stable under the conditions employed, more polar lipids were extensively reduced to the corresponding fatty alcohols. Thus when reacted with NaBH₄ for 60 min at 37°C, 94% and 64% respectively of the acyl groups in lecithin and monogalactosyl diglyceride were reduced to fatty alcohols. No discernible reduction or isomerisation of double bonds occurred during the reactions. Reductions in the reaction temperature, and in the THF content of the solvent, both resulted in slower reaction rates.The reactions with complex lipids proceded with the intermediate formation of the corresponding monoacyl (“lyso”) lipids, but the reagent showed no selectivity towards position or structure of the component ester groups.In its proposed form, the method for determining acyl thiolesters in biological tissues by their specific reduction with NaBH₄, is not satisfactory.     

Single laser three color immunofluorescence staining procedures based on energy transfer between phycoerythrin and cyanine 5.

Monoclonal antibodies specific for phycoerythrin (PE) were covalently labeled with the fluorescent dye cyanine 5 (Cy5). Excitation at 488 nm of immune complexes obtained by mixing Cy5-anti-PE with PE resulted in a 4-fold reduction of PE fluorescence measured at 565 nm and an increase of fluorescence measured at 655 nm. The observed energy transfer between PE and Cy5-anti-PE was used to develop three color immunofluorescence staining procedures for flow cytometers equipped with an Argon laser tuned at 488 nm. Mouse IgG1 monoclonal antibodies specific for cell surface antigens were cross-linked with either unlabeled or Cy5 labeled mouse IgG1 anti-PE using F(ab')2 fragments of monoclonal rat anti-mouse IgG1. PE was added to these immune complexes in sufficient amounts to saturate all PE binding sites. Cells were incubated with PE-labeled and PE/Cy5-labeled tetrameric antibody complexes together with FITC labeled antibodies and analyzed by flow cytometry. The emission from FITC, PE and PE/Cy5 could be readily separated and bright three color immunofluorescence staining of mononuclear cells from human peripheral blood and bone marrow was observed. The results of these experiments demonstrate that useful probes for single laser three color staining of cell surface antigens can be readily obtained by mixing of selected reagents. Compared to standard procedures for the covalent labeling of PE (tandem) molecules to antibodies, the non-covalent procedures described in this report provide significant advantages in terms of the amount of reagents, time and equipment required to obtain suitable reagents for three color immunofluorescence staining.     

Consequences of antisense RNA inhibition of starch branching enzyme activity on properties of potato starch

Antisense constructs containing cDNAs for potato starch branching enzyme (SBE) were introduced into potato (Solanum tuberosum L.). A population of transgenic plants were generated in which tuber SBE activity was reduced by between 5 and 98% of control values. No significant differences in amylose content or amylopectin branch length profiles of transgenic tuber starches were observed as a function of tuber SBE activity. Starches obtained from low SBE activity plants showed elevated phosphorous content. ³¹P n.m.r. analysis showed that this was due to proportionate increases in both 3- and 6-linked starch phosphates. A consistent alteration in starch gelatinisation properties was only observed when the level of SBE activity was reduced to below ˜5% of that of control values. Starches from these low SBE activity plants showed increases of up to 5 °C in d.s.c. peak temperature and viscosity onset temperature. Studies on melting of crystallites obtained from linear (1 → 4)-- -glucan oligomers suggest that an average difference of double helix length of about one glucose residue might be sufficient to account for the observed differences in gelatinisation properties. We speculate that the modification of gelatinisation properties at low SBE activities is due to a subtle alteration in amylopectin branch patterns resulting in small changes in double helix lengths within granules.     

Isolation and characterization of two Brassica napus embryo acyl-ACP thioesterase cDNA clones

Acyl-ACP thioesterases are involved in regulating chain termination of fatty acid biosynthesis in plant systems. Previously, acyl-ACP thioesterase purified from Brassica napus seed tissue has been shown to have a high preference for hydrolysing oleoyl-ACP. Here, oligonucleotides derived from B. napus oleoyl-ACP thioesterase protein sequence data have been used to isolate two acyl-ACP thioesterase clones from a B. napus embryo cDNA library. The two clones, pNL2 and pNL3, contain 1642 bp and 1523 bp respectively and differ in the length of their 3 non-coding regions. Both cDNAs contain open reading frames of 366 amino acids which encode for 42 kDa polypeptides. Mature rape thioesterase has an apparent molecular weight of 38 kDa on SDS-PAGE and these cDNAs therefore encode for precursor forms of the enzyme. This latter finding is consistent with the expected plastidial location of fatty acid synthase enzymes. Northern blot analysis shows thioesterase mRNA size to be ca. 1.6 kb and for the thioesterase genes to be highly expressed in seed tissue coincident with the most active phase of storage lipid synthesis. There is some sequence heterogeneity between the two cDNA clones, but overall they are highly homologous sharing 95.7% identity at the DNA level and 98.4% identity at the amino acid level. Some sequence heterogeneity was also observed between the deduced and directly determined thioesterase protein sequences. Consistent with the observed sequence heterogeneity was Southern blot data showing B. napus thioesterase to be encoded by a small multi-gene family.     

Biomass and elemental uptake in fertilized and unfertilizedBetula papyrifera Marsh. andPopulus grandidentata Michx.

Summary Estimates were made of the above-ground biomass and contents of N, P, K, Ca, Mg, Mn, Na, Fe, Zn, Al, and Cu in fertilized (N 448 kg/ha, P 112 kg/ha, lime 4480 kg/ha) and unfertilized white birch (Betula papyrifera Marsh.) and bigtooth aspen (Populus grandidentata Michx.). For individuals of both species, fertilization increased the average above-ground biomass increment and the N and P content increment by 150 per cent and 300 per cent, respectively, but decreased uptake of Mn and Zn. The allocation of biomass and elements differs not only between species, but within species under untreated and fertilized conditions.