The molecular biology of desiccation tolerance was investigated in the cyanobacteria with emphasis on Nostoc commune. Analysis of DNA from 41 samples of desiccated Nostoc spp. of varied age and global distribution led to the amplification of 43 independent tRNALEU(UAA) group 1 intron sequences. Phylogenetic analysis of the entire data set made it possible to define the form species Nostoc commune. The synthase (spsA) and phosphatase (sppA) genes required for the synthesis of sucrose were isolated from cyanobacterium Synechocystis sp. strain PCC 6803 and overexpressed in E. coli in two different vector constructions. Transformants had a marked increased capacity for desiccation tolerance. Sucrose synthesis was confirmed through thin layer chromatography (TLC) analysis of cell extracts from transformants. Long-term stability of DNA in desiccated Nostoc samples was demonstrated by the ability to amplify selected gene loci from samples stored dry for decades. Successful amplification in some samples was possible only after treatment with phenacylthiazolium bromide, a reagent that disrupts covalent cross-links; indicating that the DNA was modified by cross-links that occurred between reducing sugars and the primary amines on the DNA. Abundant superoxide dismutase was released following rehydration of desiccated field material N. commune CHEN after 13 years in the dry state. sodF mRNA was present in the dry material but was turned over within 15 min of rehydration. mRNA levels then rose and appeared to reach steady state levels after 3 hours and remained abundant after 24 hours of rehydration. / Master of Science

Ph.D. / Biochemistry and Molecular Biology / Selenocysteine-containing mutants of human copper chaperone for superoxide dismutase (hCCS) were constructed using intein-mediated peptide ligation. These mutants were studied with respect to their ability to transfer Cu to E,Zn superoxide dismutase (SOD1) and their Cu-binding and X-ray absorption spectroscopic (XAS) properties. Previous studies have shown that three functionally distinct polypeptide domains are present in CCS: the N-terminal domain 1 (D1, residues 1-85) contains the copper-binding MXCXXC motif, domain 2 (D2, residues 86-234) has sequence homology to residues associated with the native SOD1 dimer interface, and the C-terminal domain 3 (D3, residues 235-274) contains a CXC motif. Recent results suggest the formation of a D3- D3 cluster within a dimeric or tetrameric protein and suggest that this cluster may be an important element of the copper transfer machinery. D3 cysteine-to-selenocysteine mutants of wild-type and D1 mutants of hCCS were constructed to investigate the D3 copper cluster in more detail. These mutants display similar activity to wild-type protein. The structure of the Cu centers of selenocysteine-containing mutants as shown by Cu EXAFS is similar to that of wild-type protein, with clear indications of a Cu cluster. Cu and Se EXAFS of these constructs reveal a unique adamantane-like cluster formed between two molecules of CCS at the D3-D3 interface. These results confirm the existence of a D3-D3 copper cluster in hCCS and suggest that a unique copper cluster may exist in this protein.

Thesis (M.S.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains ix, 67 p. : ill. Includes abstract. Includes bibliographical references (p. 63-67).

Diss. (sammanfattning) Umeå : Umeå universitet, 2009. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 97-104).

Diss. (sammanfattning) Umeå : Umeå universitet, 2010. / Härtill 4 uppsatser.

In the stamen (male reproductive tissue) of petunia 'Hurrah' flowers, the occurrence of SOD (superoxide dismutase) provided an effective anti-oxidative mechanism against superoxide production. Superoxide production and SOD activities at five developmental stages showed a positive correlation. The highest superoxide production and SOD activity in different parts of the stamen (anther, filament and pollen) were at stages with high metabolic activity: (i) during growing buds (in anthers and filaments) (ii) when flowers with predehiscent anthers were fully open (in pollen). In all parts of the stamen, SOD activity was the lowest at stage five (fully open flowers with dehiscent anthers), superoxide production was also lower at this stage with the exception of the pollen. The highest SOD activity was localized in anthers with the pollen, suggesting that the filaments only have a structural support function. SOD was examined on a native PAGE with regard to the isozymes present within the stamen of five developmental stages. Three isozymes, which were identified as Mn SOD, Fe SOD and Cu/Zn SOD by reactions with inhibitors, were commonly found at five developmental stages in crude extracts of anthers, filaments and pollen. The developmental stages with stronger isozyme bands on the native PAGE were consistent with the stages with higher SOD activities, and the Mn SOD and Fe SOD isozyme bands were more intense than Cu/Zn SOD bands, suggesting the activities of Mn SOD and Fe SOD in the crude extracts were much higher than Cu/Zn SOD. SOD from 1,000 stamens of dehiscent mature flowers was partially purified using ammonium sulphate fractionation and DEAE cellulose column chromatography. The purified bound fraction contained only one SOD isozyme on a native PAGE, which was shown to be a Mn SOD, as it is sensitive to neither hydrogen peroxide nor cyanide. The specific activity of the purified SOD was 66.5 U/mg and the yield of total activity was 3.0%. The progress of enzyme purification was monitored using SDS-PAGE and the bound fraction contained two major polypeptide bands. The purified enzyme activity was optimal in the range of neutral pH, but it was the highest at pH 7.8. Through incubation at various pH levels for 24 hours, favourable stability of the purified fraction was confirmed around a pH range of 7 to 8.5. The purified enzyme retained 87% of its initial activity at -20 ? after one month of storage, but at 4 ? only 38% of the initial activity remained after the same period of storage.

O objetivo do estudo foi estimar a biodisponibilidade de duas fontes de cobre: orgânica (cobre metionina) e inorgânica (sulfato de cobre) na dieta de cordeiros. O experimento foi conduzindo na FZEA USP de Pirassununga utilizando 40 cordeiros da raça Merino X Texel, que foram distribuídos aleatoriamente em cinco grupos, e submetidos a cinco tratamentos, totalizando oito animais por tratamento: Tratamento 0: Dieta controle sem adição de Cu; Tratamento 1: 10 mg de Cu/Kg de MS na forma de sulfato de Cu; Tratamento 2: 30g de Cu/Kg de MS na forma de sulfato de Cu; Tratamento 3: 10 mg de cu/kg de MS na forma de cobre metionina; Tratamento 4: 30 mg de cu/kg de MS na forma de cobre metionina. Foram feitas biópsias do fígado dos animais no tempo zero para análise de cobre e colhidas amostras de sangue nos dias 0, 28, 56 e 84 dias para determinação de Cu sérico, atividade de ceruloplasmina e enzimas de função hepática. Ao final do experimento, os animais foram abatidos para colheita de amostras de fígado, músculo e rim, para determinação dos teores de Cu e da enzima superóxido dismutase (SOD). Nos últimos dez dias do experimento foi realizado um balanço metabólico de cobre. A biodisponibilidade foi calculada pela técnica \"slope ratio\", utilizando como parâmetros a concentração de cobre no fígado. Não houve diferença (P>0,05) no desempenho dos animais (peso vivo e ganho de peso) entre os tratamentos. A concentração sérica de AST e ALT permaneceu abaixo dos níveis de intoxicação em todos os tratamentos, durante todo o período. A atividade da ceruloplasmina não diferiu entre os tratamentos (P>0,05). O teor de cobre no soro, na biópsia do fígado e no músculo não foi diferente (P>0,05) entre os tratamentos. Entretanto, a concentração do mineral no fígado dos animais suplementados (284,28 mg/kg) foi maior (P<0,05), quando comparados ao grupo controle (168,01 mg/kg), assim como o Cu-met 30 mg/kg (341,29 mg/kg) foi superior (P<0,05) ao de 10mg/kg MS (263,02 mg/kg). A atividade da SOD nos animais suplementados (µmol/mg prot foi superior à do grupo controle. Nos rins o teor de cobre foi superior nos animais que receberam 30mg/kg de MS de Cu-met (6,65 mg/kg) em relação aos que receberam 10 mg/kg de MS da mesma fonte (3,86 mg/kg). A absorção e a retenção aparentes do cobre foram maiores para a fonte inorgânica, comparada com a orgânica. A biodisponibilidade do cobre determinada pela concentração de cobre no fígado, utilizando a técnica do \"slope ratio\", considerando o CuSO4 como padrão (100%), apresentou disponibilidade de 150,64% para o Cu-met. / The study was conducted to estimate the relative bioavailability of two sources of supplemental copper: organic (copper methionine) and inorganic (copper sulfate) in the diet of lambs, by analyzing the concentration of copper and enzymes in the liver and metabolic balance calculation, using 40 lambs breed Merino X Texel, which was fed three concentrations of copper (basal + two additions) in two sources, which were randomly allotted to five groups, and subjected to five treatments: treatment 0: control (diet without addition of Cu); treatment 1: (diet with 10 mg Cu/Kg DM of CuSO4); Treatment 2: (diet with 30 g Cu/Kg DM of CuSO4); Treatment 3: (diet with 10 mg Cu/kg DM of copper methionine; Treatment 4: (diet with 30 mg cu/kg DM of copper methionine). Liver biopsies were made on 0 d. Blood samples were taken via the jugular vein on 0, 28, 56 and 84 d to determine serum Cu and serum ceruloplasmin and liver transaminases (AST, ALT) concentrations. The animals were slaughtered and samples of liver, kidney and muscle were taken for the determination of the levels of Cu and superoxide dismutase activity. In the last ten days of the experiment a metabolic balance of copper was conducted. The bioavailability was calculated by the \"slope ratio\" technique, using the concentration of copper in the liver as parameter. There was no difference (P >0.05) on animal performance (live weight and weight gain) among treatments. The serum AST and ALT levels remained below poisoning in all treatments during the period. The ceruloplasmin activity did not differ between treatments (P>0.05). The copper content in biopsy, serum and muscle was not different (P>0.05) between treatments. However, the mineral concentration in the liver of animals fed (284.28 mg/kg) was higher (P <0.05) when compared to the control group (168.01 mg/kg ) and 30 Cu -met mg/kg (341.29 mg/kg) was higher (P <0.05) at 10mg/kg MS (263.02 mg/kg). The SOD activity in the supplemented animals (mmol/mg prot) was superior to the control group. Copper in Kidneys was higher in animals that received 30mg/kg MS meth-Cu (6.65mg/kg) compared those receiving 10 mg/kg DM from the same source (3.86 mg/kg). Apparent absorption and retention of copper were higher for inorganic source, compared with the organic. The bioavailability determined by the concentration of copper in the copper liver, using the technique of \"slope ratio\", considering CuSO4 as standard ( 100% ) presented availability of 150.64 % for Cu-met.

Cu/Zn Superoxide Dismutase (SOD1), is a ubiquitous antioxidant enzyme with several emerging roles outside of its canonical function. SOD1 is also emerging in central roles in cancer and neurodegenerative pathologies. Little is known about SOD1 regulation, particularly at a post-translational level. Post-translational modifications (PTMs) play an important role in enabling proteins to rapidly respond to their environment. Therefore, identifying specific PTMs involved in protein regulation represents a powerful opportunity to interfere with any associated pathologies. This work employs proteomics to identify mechanisms of post-translation regulation on cell survival signaling proteins. We focused on SOD1, which protects cells from oxidative stress. We found that acylation of K122 on SOD1, while not impacting SOD1 catalytic activity, suppressed the ability of SOD1 to inhibit mitochondrial metabolism at respiratory complex I. We found that deacylase depletion increased K122 acylation on SOD1, which blocked suppression of respiration in a K122-dependent manner. In addition, we found that acyl-mimicking mutations at K122 decreased SOD1 accumulation in mitochondria, initially hinting that SOD1 may inhibit respiration directly within the intermembrane space (IMS). However, surprisingly, we found that forcing the K122 acyl mutants into the mitochondria with an IMS-targeting tag did not recover their ability to suppress respiration. Moreover, we found that suppressing or boosting respiration levels toggled SOD1 in or out of the mitochondria, respectively. These findings place SOD1-mediated inhibition of respiration upstream of its mitochondrial localization. Interestingly, we also found that K122 acyl mutants were sufficient to prevent mitochondrial accumulation of the G93A SOD1 clinical mutant. We observed increased autophagic activity in G93A expressing cells compared to WT or G93A/K122-acyl mimic double mutants, and found that this double mutant was just as prone to aggregate as G93A SOD1—suggesting that SOD1 aggregation is more toxic when in the mitochondria. We observed increased protein turnover rates in cells expressing SOD1 G93A, in support of increased autophagy. Lastly, deletion-rescue experiments show that a respiration-defective mutant of SOD1 is also impaired in its ability to rescue cells from toxicity caused by SOD1 deletion. Together, these data suggest a new interplay between SOD1 acylation, metabolic regulation, SOD1 aggregate toxicity, and SOD1-mediated cell survival.

Copper is an essential cofactor of two mitochondrial enzymes: cytochrome c oxidase (COX) and the mitochondrial localized fraction of Cu-Zn superoxide dismutase (Sod1p). Copper incorporation into these enzymes is facilitated by a growing number of metallochaperone proteins. Here we describe two novel copper chaperones of COX, Cmc1 and Cmc2. In Saccharomyces cerevisiae, both Cmc1 and Cmc2 localize to the mitochondrial inner membrane facing the intermembrane space. Cmc1 and Cmc2 are essential for full expression of COX and cellular respiration, contain a twin Cx9C domain, and are conserved from yeast to humans. Additionally, the presence or absence of these proteins not only determines full assembly of functional COX but also affects metallation of Sod1 suggesting these proteins might play a role on co-modulation of copper transfer to COX and Sod1. CMC1 overexpression does not rescue the respiratory defect of cmc2 mutants or vise versa. However, Cmc2 physically interacts with Cmc1 and the absence of Cmc2 induces a 5-fold increase in Cmc1 accumulation in the mitochondrial membranes. We conclude that Cmc1 and Cmc2 have cooperative but non-overlapping functions in cytochrome c oxidase biogenesis.