Fatty acid ω-hydroxylases from <Emphasis Type="Italic">Solanum tuberosum</Emphasis>

Key message

Potato StCYP86A33 complements the Arabidopsis AtCYP86A1 mutant, horst - 1.

Abstract

Suberin is a cell-wall polymer that comprises both phenolic and aliphatic components found in specialized plant cells. Aliphatic suberin is characterized by bi-functional fatty acids, typically ω-hydroxy fatty acids and α,ω-dioic acids, which are linked via glycerol to form a three-dimensional polymer network. In potato (Solanum tuberosum L.), over 65 % of aliphatics are either ω-hydroxy fatty acids or α,ω-dioic acids. Since the biosynthesis of α,ω-dioic acids proceeds sequentially through ω-hydroxy fatty acids, the formation of ω-hydroxy fatty acids represents a significant metabolic commitment during suberin deposition. Four different plant cytochrome P450 subfamilies catalyze ω-hydroxylation, namely, 86A, 86B, 94A, and 704B; though to date, only a few members have been functionally characterized. In potato, CYP86A33 has been identified and implicated in suberin biosynthesis through reverse genetics (RNAi); however, attempts to express the CYP86A33 protein and characterize its catalytic function have been unsuccessful. Herein, we describe eight fatty acid ω-hydroxylase genes (three CYP86As, one CYP86B, three CYP94As, and a CYP704B) from potato and demonstrate their tissue expression. We also complement the Arabidopsis cyp86A1 mutant horst-1 using StCYP86A33 under the control of the Arabidopsis AtCYP86A1 promoter. Furthermore, we provide preliminary analysis of the StCYP86A33 promoter using a hairy root transformation system to monitor pStCYP86A33::GUS expression constructs. These data confirm the functional role of StCYP86A33 as a fatty acid ω-hydroxylase, and demonstrate the utility of hairy roots in the study of root-specific genes.

     

Regulation of Hsp90 ATPase activity by tetratricopeptide repeat (TPR)-domain co-chaperones.

The in vivo function of the heat shock protein 90 (Hsp90) molecular chaperone is dependent on the binding and hydrolysis of ATP, and on interactions with a variety of co-chaperones containing tetratricopeptide repeat (TPR) domains. We have now analysed the interaction of the yeast TPR-domain co-chaperones Sti1 and Cpr6 with yeast Hsp90 by isothermal titration calorimetry, circular dichroism spectroscopy and analytical ultracentrifugation, and determined the effect of their binding on the inherent ATPase activity of Hsp90. Sti1 and Cpr6 both bind with sub-micromolar affinity, with Sti1 binding accompanied by a large conformational change. Two co-chaperone molecules bind per Hsp90 dimer, and Sti1 itself is found to be a dimer in free solution. The inherent ATPase activity of Hsp90 is completely inhibited by binding of Sti1, but is not affected by Cpr6, although Cpr6 can reactivate the ATPase activity by displacing Sti1 from Hsp90. Bound Sti1 makes direct contact with, and blocks access to the ATP-binding site in the N-terminal domain of Hsp90. These results reveal an important role for TPR-domain co-chaperones as regulators of the ATPase activity of Hsp90, showing that the ATP-dependent step in Hsp90-mediated protein folding occurs after the binding of the folding client protein, and suggesting that ATP hydrolysis triggers client-protein release.     

Regulation of Hsp90 ATPase activity by the co-chaperone Cdc37p/p50cdc37

In vivo activation of client proteins by Hsp90 depends on its ATPase-coupled conformational cycle and on interaction with a variety of co-chaperone proteins. For some client proteins the co-chaperone Sti1/Hop/p60 acts as a "scaffold," recruiting Hsp70 and the bound client to Hsp90 early in the cycle and suppressing ATP turnover by Hsp90 during the loading phase. Recruitment of protein kinase clients to the Hsp90 complex appears to involve a specialized co-chaperone, Cdc37p/p50(cdc37), whose binding to Hsp90 is mutually exclusive of Sti1/Hop/p60. We now show that Cdc37p/p50(cdc37), like Sti1/Hop/p60, also suppresses ATP turnover by Hsp90 supporting the idea that client protein loading to Hsp90 requires a "relaxed" ADP-bound conformation. Like Sti1/Hop/p60, Cdc37p/p50(cdc37) binds to Hsp90 as a dimer, and the suppressed ATPase activity of Hsp90 is restored when Cdc37p/p50(cdc37) is displaced by the immunophilin co-chaperone Cpr6/Cyp40. However, unlike Sti1/Hop/p60, which can displace geldanamycin upon binding to Hsp90, Cdc37p/p50(cdc37) forms a stable complex with geldanamycin-bound Hsp90 and may be sequestered in geldanamycin-inhibited Hsp90 complexes in vivo.     

Towards a comprehensive barcode library for arctic life - Ephemeroptera, Plecoptera, and Trichoptera of Churchill, Manitoba, Canada

Background

In order to understand the role of herbivores in trophic webs, it is essential to know what they feed on. Diet analysis is, however, a challenge in many small herbivores with a secretive life style. In this paper, we compare novel (high-throughput pyrosequencing) DNA barcoding technology for plant mixture with traditional microhistological method. We analysed stomach contents of two ecologically important subarctic vole species, Microtus oeconomus and Myodes rufocanus, with the two methods. DNA barcoding was conducted using the P6-loop of the chloroplast trnL (UAA) intron.

Results

Although the identified plant taxa in the diets matched relatively well between the two methods, DNA barcoding gave by far taxonomically more detailed results. Quantitative comparison of results was difficult, mainly due to low taxonomic resolution of the microhistological method, which also in part explained discrepancies between the methods. Other discrepancies were likely due to biases mostly in the microhistological analysis.

Conclusion

We conclude that DNA barcoding opens up for new possibilities in the study of plant-herbivore interactions, giving a detailed and relatively unbiased picture of food utilization of herbivores.     

Characterization of a partially denatured state of a protein by two-dimensional NMR: reduction of the hydrophobic interactions in ubiquitin

A stable, partially structured state of ubiquitin, the A-state, is formed at pH 2.0 in 60% methanol/40% water at 298 K. Detailed characterization of the structure of this state has been carried out by 2D NMR spectroscopy. Assignment of slowly exchanging amide resonances protected from the solvent in the native and A-state shows that gross structural reorganization of the protein has not occurred and that the A-state contains a subset of the interactions present in the native state (N-state). Vicinal coupling constants and NOESY data show the presence of the first two strands of the five-strand beta-sheet that is present in the native protein and part of the third beta-strand. The hydrophobic face of the beta-sheet in the A-state is covered by a partially structured alpha-helix, tentatively assigned to residues 24-34, that is considerably more flexible than the alpha-helix in the N-state. There is evidence for some fixed side-chain--side-chain interactions between these two units of structure. The turn-rich area of the protein, which contains seven reverse turns and a short piece of 3(10) helix, does not appear to be structured in the A-state and is approaching random coil.     

Deviations from Michaelis-Menten kinetics. Computation of the probabilities of obtaining complex curves from simple kinetic schemes.

1. It is possible to calculate the intrinsic probability associated with any curve shape that is allowed for rational functions of given degree when the coefficients are independent or dependent random variables with known probability distributions. 2. Computations of such probabilities are described when the coefficients of the rational function are generated according to several probability distribution functions and in particular when rate constants are varied randomly for several simple model mechanisms. 3. It is concluded that each molecular mechanism is associated with a specific set of curve-shape probabilities, and this could be of value in discriminating between model mechanisms. 4. It is shown how a computer program can be used to estimate the probability of new complexities such as extra inflexions and turning points as the degree of rate equations increases. 5. The probability of 3 : 3 rate equations giving 2 : 2 curve shapes is discussed for unrestricted coefficients and also for the substrate-modifier mechanisms. 6. The probability associated with the numerical values of coefficients in rate equations is also calculated for this mechanism, and a possible method for determining the approximate magnitude of product-release steps is given. 7. The computer programs used in the computations have been deposited as Supplement SUP 50113 (21 pages) with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem, J. (1978) 169, 5.     

Tono Reservoir fishery contribution to poverty reduction among fishers in northern Ghana

Fishery characteristics and livelihood status of fishers at Tono Reservoir, Ghana, were investigated between January 2015 and June 2016. Data on fisher demography, fishing gears, fishing methods, perceptions of the state of fish stocks, management practices, income and consumption of fishers were obtained through structured interviews. Censuses of fishers and fishing gears were conducted through direct observation and counts. The population of fishers was 950 and the majority (74%) of the sampled respondents fell within the ages of 24–41 years. Gillnet, cast net, trap and hook and line were the four main gears utilised. Illegal methods of fishing observed included the use of mosquito nets (nets with mesh <1.0 cm) and the use of brewer’s waste (pito mash) as bait. Brycinus nurse, Synodontis spp., Parailia spiniserrata and Chrysichthys spp. were perceived to have disappeared from the reservoir. The fishers were unaware of the existence of any fisheries regulations, hence there was no adherence to management practices. Their daily income was derived mainly from fishing. The incidence of poverty among fishers was low (8%). The Tono Reservoir has a great potential for supporting livelihood if it is properly managed.     

On the statistical evaluation of adherence assays

Parametric (unpaired t-test) and non-parametric (Mann-Whitney U-test) methods have been used in the evaluation of adherence assays on the non-antibiotic antimicrobial agent, Taurolin. In all but one case, where the anti-adherence effect was known to be marginal, both statistical methods gave similar results although there were some minor differences in the levels of significance achieved. The effect of the agent on the deviation of adherence data from normality was quantified by calculation of the skewness coefficient for each data set. A significant anti-adherence effect appears to result in a decrease in the skewness of the adherence assay data. It was concluded that either parametric or non-parametric statistical evaluation of adherence assay data is valid for large numbers of observations. In future studies of this type it is suggested that attention should also be given to the effect of the anti-adherence agent on the deviation of adherence data from normality as denoted by the skewness coefficient.     

Non‐verbal interaction of Anglo‐Canadian,Jewish‐Canadian,and French‐Canadian physicians with their young,middle‐aged,and elderly patients

This study investigates the role that non‐verbal communication plays in establishing and maintaining rapport among physicians and their young, middle‐aged, and elderly patients.

Eleven physicians with a variety of patients were studied in the project in Montreal. Five physicians were Anglo‐Canadian, three were respectively Jewish‐Canadian and French‐Canadian. Three additional physicians were part of a pilot project.

Patient‐doctor interviews were videotaped by means of a stationary camera fitted with a wide‐angle lens placed in the doctors’ offices. Physical examinations were not videotaped. After each session, patients filled out a questionnaire elucidating the success of the interview.