Import-associated translational inhibition: novel in vivo evidence for cotranslational protein import into dictyostelium discoideum mitochondria

To investigate protein import into the mitochondria of Dictyostelium discoideum, green fluorescent protein (GFP) was fused as a reporter protein either to variable lengths of the N-terminal region of chaperonin 60 (the first 23, 40, 80, 97, and 150 amino acids) or to the mitochondrial targeting sequence of DNA topoisomerase II. The fusion proteins were expressed in AX2 cells under the actin-15 promoter. Fluorescence images of GFP transformants confirmed that Dictyostelium chaperonin 60 is a mitochondrial protein. The level of the mitochondrially targeted GFP fusion proteins was unexpectedly much lower than the nontargeted (cytoplasmic) forms. The distinction between targeted and nontargeted protein activities was investigated at both the transcriptional and translational levels in vivo. We found that targeting GFP to the mitochondria results in reduced levels of the fusion protein even though transcription of the fusion gene and the stability of the protein are unaffected. [³⁵S]methionine labeling and GFP immunoprecipitation confirmed that mitochondrially targeted GFP is translated at much slower rates than nontargeted GFP. The results indicate a novel phenomenon, import-associated translational inhibition, whereby protein import into the mitochondria limits the rate of translation. The simplest explanation for this is that import of the GFP fusion proteins occurs cotranslationally, i.e., protein synthesis and import into mitochondria are coupled events. Consistent with cotranslational import, Northern analysis showed that the GFP mRNA is associated with isolated mitochondria. This association occurred regardless of whether the GFP was fused to a mitochondrial leader peptide. However, the presence of an import-competent leader peptide stabilized the mRNA-mitochondria association, rendering it more resistant to extensive EDTA washing. In contrast with GFP, the mRNA of another test protein, aequorin, did not associate with the mitochondria, and its translation was unaffected by import of the encoded polypeptide into the mitochondria.

Bioinformatic and expression analyses of genes mediating zinc homeostasis in Nostoc punctiforme

Zinc homeostasis was investigated in Nostoc punctiforme. Cell tolerance to Zn²⁺ over 14 days showed that ZnCl₂ levels above 22 µM significantly reduced cell viability. After 3 days in 22 µM ZnCl₂, ca. 12% of the Zn²⁺ was in an EDTA-resistant component, suggesting an intracellular localization. Zinquin fluorescence was detected within cells exposed to concentrations up to 37 µM relative to 0 µM treatment. Radiolabeled ⁶⁵Zn showed Zn²⁺ uptake increased over a 3-day period, while efflux occurred more rapidly within a 3-h time period. Four putative genes involved in Zn2+ uptake and efflux in N. punctiforme were identified: (i) the predicted Co/Zn/Cd cation transporter, putative CDF; (ii) the predicted divalent heavy-metal cation transporter, putative Zip; (iii) the ATPase component and Fe/Zn uptake regulation protein, putative Fur; and (iv) an ABC-type Mn/Zn transport system, putative zinc ZnuC, ZnuABC system component. Quantitative real-time PCR indicated the responsiveness of all four genes to 22 µM ZnCl₂ within 3 h, followed by a reduction to below basal levels after 24 h by putative ZIP, ZnuC, and Fur and a reduction to below basal level after 72 h by putative CDF efflux gene. These results demonstrate differential regulation of zinc transporters over time, indicating a role for them in zinc homeostasis in N. punctiforme.

Aqueous/micellar peroxyoxalate chemiluminescent detection for ion chromatography

This thesis covers the development of the traditionally fluorescent bis(8-quinolinol-5-sulfonic acid) magnesium (II) fluorophore as a chemiluminescent emitter. A brief description of luminescence spectroscopy and its application to analytical chemistry lays the foundation to the discussion of the results obtained herein. This includes the synthesis and identification of two so called ‘water soluble’ aryl oxamides 2,2’-oxalyl-bis(trifluoromethanesulfonyl) imino] ethylene-bis(N- methylpyridinium) trifluoromethane sulfonate (PETQ) and 2,2’-oxalyl-bis(trifluoromethanesulfonyl) imino]ethylene-bis(N-pyridinium) chloride (PETH), previously developed for the US navy as a possible emergency light source, yet the synthetic methodology were incomplete. The inconsistencies of the synthetic methods for PETQ and PETH were overcome with yields satisfactory for their preliminary analytical evaluation. The evaluation of these aryl oxamides, including 4,4’-oxalyI- bis[(trifluoromethanesulfonyl) imino]ethylene-bis(l-methyM-benzylpiperidinium) trifluoromethanesulfonate (BPTQ), 4,4’-oxalyl-bis [(trifluoromethylsulfonyl)imino] ethylene-bis(N-methylmorpholinium)trifluoromethanesulfonate (METQ) and the oxalate bis(2,4,6-trichlorophenyl) oxalate (TCPO) were performed with the peroxyoxalate chemiluminescent reaction using bis(8-quinolinol-5-sulfonic acid) magnesium (II) as the fluorophore. A univariate optimisation of this system resulted in 0,0082 mol 1-1 the detection limit of magnesium in the absence of cationic surfactants and 0.0041 mol 1-1 in their presence for the majority of these compounds. The oxamides were found to be insoluble in water with long ulrasonication periods required to dissolve the compound, with solvents such as acetonitrile preferred. The determination of other chemiluminescent metal-8HQS chelates to replace magnesium -8HQS in the peroxyoxalate were limited to Al (III), Cd (II), Ca (II), In (II) and Zn (II), unfortunately these metals all possessed poorer detection limits than those obtained using magnesium The base reaction conditions used for the flow injection system with chemiluminescent detection were transferred to an ion chromatographic configuration for the separation of magnesium from other cations on an exchange column. After a univariate and simplex optimisation of these conditions, the detection limit of magnesium was found to be 0.0411 mol 1-1 which was less than the limits that could be achieved with fluorescent detection, The further development of this reaction to incorporate the displacement of magnesium from Mg-EDTA by other metals that possessed a higher conditional stability constant than magnesium also proved to be problematic with interferences from not only EDTA but from the eluant (lactic acid) from the cation column. Using this system the detection limits of the displacing metals were found to be in the order of 10 mg 1-1 which was substantially less that what was observed when exactly the same configuration was used with fluorescent detection. The final component of the thesis entails the discussion of the background emission that results from the reaction of oxamides/oxalates with hydrogen peroxide. A detailed investigation into the reaction of TCPO and hydrogen peroxide in the presence of various additives, such as imidazole , heavy atoms and triethylamine illustrated the existence of a further intermediate in fee mechanism for this reaction. The species responsible for this emission was attributed to the degradation product 2,4,6-trichlorophenyi of TCPO, which was supported by the non-existent background present with the oxamides that do not contain this degradation product.

Investigating ionic effects applied to water based organocatalysed aldol reactions

Saturated aqueous solutions of various common salts were examined for their effect on aqueous aldol reactions catalysted by a highly active C₂-symmetric diprolinamide organocatalyst developed in our laboratory. With respect to the aldol reaction between cyclohexanone and 4-nitrobenzaldehyde, deionised water was always a superior medium to salt solutions though some correlation to increasing anion size and depression in enantiomeric excess could be observed. Additionally, the complete inhibition of catalyst activity observed when employing tap water could be alleviated by the inclusion of ethylenediaminetetraacetate (EDTA) into the aqueous media prior to reaction initiation. Extension of these reaction conditions demonstrated that these ionic effects vary on a case-to-case basis depending on the ketone/aldehyde combination.

Physiological metal uptake by Nostoc punctiforme

Trace metals are required for many cellular processes. The acquisition of trace elements from the environment includes a rapid adsorption of metals to the cell surface, followed by a slower internalization. We investigated the uptake of the trace elements Co²⁺, Cu²⁺, Mn²⁺, Ni²⁺, and Zn²⁺ and the non-essential divalent cation Cd²⁺ in the cyanobacterium Nostoc punctiforme. For each metal, a dose response study based on cell viability showed that the highest non-toxic concentrations were: 0.5 μM Cd²⁺, 2 μM Co²⁺, 0.5 μM Cu²⁺, 500 μM Mn²⁺, 1 μM Ni²⁺, and 18 μM Zn²⁺. Cells exposed to these non-toxic concentrations with combinations of Zn²⁺ and Cd²⁺, Zn²⁺ and Co²⁺, Zn²⁺ and Cu²⁺ or Zn²⁺ and Ni²⁺, had reduced growth in comparison to controls. Cells exposed to metal combinations with the addition of 500 μM Mn²⁺ showed similar growth compared to the untreated controls. Metal levels were measured after one and 72 h for whole cells and absorbed (EDTA-resistant) fractions and used to calculate differential uptake rates for each metal. The differences in binding and internalisation between different metals indicate different uptake processes exist for each metal. For each metal, competitive uptake experiments using ⁶⁵Zn showed that after 72 h of exposure Zn²⁺ uptake was reduced by most metals particularly 0.5 μM Cd²⁺, while 2 μM Co²⁺ increased Zn²⁺ uptake. This study demonstrates that N. punctiforme discriminates between different metals and favourably substitutes their uptake to avoid the toxic effects of particular metals.

Cotranslational assembly of the yeast SET1C histone methyltransferase complex

 While probing the role of RNA for the function of SET1C/COMPASS histone methyltransferase, we identified SET1RC (SET1 mRNA-associated complex), a complex that contains SET1 mRNA and Set1, Swd1, Spp1 and Shg1, four of the eight polypeptides that constitute SET1C. Characterization of SET1RC showed that SET1 mRNA binding did not require associated Swd1, Spp1 and Shg1 proteins or RNA recognition motifs present in Set1. RNA binding was not observed when Set1 protein and SET1 mRNA were derived from independent genes or when SET1 transcripts were restricted to the nucleus. Importantly, the protein-RNA interaction was sensitive to EDTA, to the translation elongation inhibitor puromycin and to the inhibition of translation initiation in prt1-1 mutants. Taken together, our results support the idea that SET1 mRNA binding was dependent on translation and that SET1RC assembled on nascent Set1 in a cotranslational manner. Moreover, we show that cellular accumulation of Set1 is limited by the availability of certain SET1C components, such as Swd1 and Swd3, and suggest that cotranslational protein interactions may exert an effect in the protection of nascent Set1 from degradation.

The significant effect of the phase composition on the oxygen reduction reaction activity of a layered oxide cathode

Layered oxides of Sr4Fe4Co2O13 (SFC2) which contains alternating perovskite oxide octahedral and polyhedral oxide double layers are attractive for their mixed ionic and electronic conducting and oxygen reduction reaction properties. In this work, we used the EDTA–citrate synthesis technique to prepare SFC2 and vary the calcination temperature between 900 and 1100 _C to obtain SFC2, containing different phase content of perovskite (denoted as SFC-P) and (Fe,Co) layered oxide phases (SFC-L). Rietveld refinements show that the SFC-P phase content increased from _39 wt% to _50 wt% and _61 wt% as the calcination temperature increased from 900 _C (SFC2-900) to 1000 _C (SFC2-1000) and 1050 _C (SFC2-1050). At 1100 _C (SFC2-1100), SFC-P became the dominant phase. The oxygen transport properties (e.g. oxygen chemical diffusion coefficient and oxygen permeability), electrical conductivity and oxygen reduction reaction activity is enhanced in the order of SFC2-1000, SFC2-1100 and SFC2-1050. The trend established here therefore negates the hypothesis that the perovskite phase content correlates with the oxygen transport property enhancement. The results suggest instead that there is an optimum composition value (e.g. 61 wt% of SFC-L for SFC2-1050 in this work) on which synergistic effects take place between the SFC-P and SFC-L phase.

Glomerular hypertrophy in subjects with low nephron number: contributions of sex, body size and race

Background. We have shown that low nephron number (Nglom) is a strong determinant of individual glomerular volume (IGV) in male Americans. However, whether the same pattern is present in female Americans remains unclear. The contributions of body surface area (BSA) and race to IGV in the context of Nglom also require further evaluation. Methods. Kidneys without overt renal disease were collected at autopsy in Mississippi, USA. The extremes of female Nglom were used to define high and low N glom for both sexes. Nglom and IGV were estimated by design-based stereology. A total of 24 African and Caucasian American females (n = 12 per race; 6 per Nglom extreme) were included. These subjects were subsequently matched to 24 comparable males by age and Nglom and to 18 additional males by age, Nglom and BSA. Results. IGV average and variance were very similar in female African and Caucasian Americans with high and low Nglom. Males with low Nglom from both races showed greater IGV average and variance than comparable females matched by age and Nglom. These differences in IGV between sexes were not observed in Caucasian Americans with low Nglom that were matched by age, Nglom and BSA. In contrast, glomeruli from African Americans were larger than those from Caucasian Americans, especially in subjects with high Nglom. Conclusions. While female Americans with low Nglom did not show glomerular hypertrophy, comparable males with low Nglom showed marked glomerular hypertrophy that was closely associated with high BSA. Glomerular size in African Americans may be confounded by multiple additional factors. © The Author 2014. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

Effect of flour processing on the quality characteristics of a soy-based beverage

Four treatments (roasting, germination, autoclaving and an application of 0.5% EDTA+0.5% sodium hydroxide) were used to reduce the beany flavour of soya beans to produce a soy-based beverage. While germination significantly increased the protein level as compared to the other treatments, the maximum reduction of the beany flavour was achieved by the 0.5% EDTA+0.5% sodium hydroxide application. The soya beans that underwent this treatment were used during the second phase for optimized beverage formulation. During the second phase, a beverage was prepared according to different formulations and analysed for chemical composition and total viable count during a two-month storage period. During storage, the beverage samples exhibited variations in several parameters. The acidity, reducing sugars and total sugars increased, while the ascorbic acid, total soluble solids and pH decreased. Overall, chemical and microbial analyses showed the stability of the product during the storage period.