Functional genomic and molecular analyses of transcripts related to glycerol synthesis and trafficking in rainbow smelt (Osmerus mordax) using cold temperature-induced models of glycerol production

The rainbow smelt (Osmerus mordax) is an anadromous teleost that produces type II antifreeze protein (AFP) and accumulates modest urea and high glycerol levels in plasma and tissues as adaptive cryoprotectant mechanisms in sub-zero temperatures. It is known that glyceroneogenesis occurs in liver via a branch in glycolysis and gluconeogenesis and is activated by low temperature; however, the precise mechanisms of glycerol synthesis and trafficking in smelt remain to be elucidated. The objective of this thesis was to provide further insight using functional genomic techniques [e.g. suppression subtractive hybridization (SSH) cDNA library construction, microarray analyses] and molecular analyses [e.g. cloning, quantitative reverse transcription - polymerase chain reaction (QPCR)]. Novel molecular mechanisms related to glyceroneogenesis were deciphered by comparing the transcript expression profiles of glycerol (cold temperature) and non-glycerol (warm temperature) accumulating hepatocytes (Chapter 2) and livers from intact smelt (Chapter 3). Briefly, glycerol synthesis can be initiated from both amino acids and carbohydrate; however carbohydrate appears to be the preferred source when it is readily available. In glycerol accumulating hepatocytes, levels of the hepatic glucose transporter (GLUT2) plummeted and transcript levels of a suite of genes (PEPCK, MDH2, AAT2, GDH and AQP9) associated with the mobilization of amino acids to fuel glycerol synthesis were all transiently higher. In contrast, in glycerol accumulating livers from intact smelt, glycerol synthesis was primarily fuelled by glycogen degradation with higher PGM and PFK (glycolysis) transcript levels. Whether initiated from amino acids or carbohydrate, there were common metabolic underpinnings. Increased PDK2 (an inhibitor of PDH) transcript levels would direct pyruvate derived from amino acids and / or DHAP derived from G6P to glycerol as opposed to oxidation via the citric acid cycle. Robust LIPL (triglyceride catabolism) transcript levels would provide free fatty acids that could be oxidized to fuel ATP synthesis. Increased cGPDH (glyceroneogenesis) transcript levels were not required for increased glycerol production, suggesting that regulation is more likely by post-translational modification. Finally, levels of a transcript potentially encoding glycerol-3-phosphatase, an enzyme not yet characterized in any vertebrate species, were transiently higher. These comparisons also led to the novel discoveries that increased G6Pase (glucose synthesis) and increased GS (glutamine synthesis) transcript levels were part of the low temperature response in smelt. Glucose may provide increased colligative protection against freezing; whereas glutamine could serve to store nitrogen released from amino acid catabolism in a non-toxic form and / or be used to synthesize urea via purine synthesis-uricolysis. Novel key aspects of cryoprotectant osmolyte (glycerol and urea) trafficking were elucidated by cloning and characterizing three aquaglyceroporin (GLP)-encoding genes from smelt at the gene and cDNA levels in Chapter 4. GLPs are integral membrane proteins that facilitate passive movement of water, glycerol and urea across cellular membranes. The highlight was the discovery that AQP10ba transcript levels always increase in posterior kidney only at low temperature. This AQP10b gene paralogue may have evolved to aid in the reabsorption of urea from the proximal tubule. This research has contributed significantly to a general understanding of the cold adaptation response in smelt, and more specifically to the development of a working scenario for the mechanisms involved in glycerol synthesis and trafficking in this species.

(Table 2) Concentrations and D/L values for three amino acids in bulk foraminifera from ODP Sites 182-1126 and 182-1127

A number of studies have shown that methanogens are active in the presence of sulfate under some conditions. This phenomenon is especially exemplified in carbonate sediments of the southern Australian continental margin. Three sites cored during Ocean Drilling Program (ODP) Leg 182 in the Great Australian Bight have high concentrations of microbially-generated methane and hydrogen sulfide throughout almost 500 m of sediments. In these cores, the sulfate-reducing and methanogenic zones overlap completely; that is, the usual sulfate-methane transition zone is absent. Amino acid racemization data show that the gassy sediments consist of younger carbonates than the low-gas sites. High concentrations of the reduced gases also occur in two ODP sites on the margin of the Bahamas platform, both of which have similar sedimentary conditions to those of the high-gas sites of Leg 182. Co-generation of these reduced gases results from an unusual combination of conditions, including: (1) a thick Quaternary sequence of iron-poor carbonate sediments, (2) a sub-seafloor brine, and (3) moderate amounts of organic carbon. The probable explanation for the co-generation of hydrogen sulfide and methane in all these sites, as well as in other reported environments, is that methanogens are utilizing non-competitive substrates to produce methane within the sulfate-reducing zone. Taken together, these results form the basis of a new model for sulfate reduction and methanogenesis in marine sediments. The biogeochemical end-members of the model are: (1) minimal sulfate reduction, (2) complete sulfate reduction followed by methanogenesis, and (3) overlapping sulfate reduction and methanogenesis with no transition zone.

Granulometry, geochemistry, amino acids, radiocarbon ages and paleomagnetics of samples from Heidemann Valley

A Pliocene (2.6-3.5 Ma) age is determined from glacial sediments studied in a 20m long, 4 m deep trench excavated in Heidemann Valley, Vestfold Hills, East Antarctica. The age determination is based on a combined study of amino acid racemization, diatoms, foraminifera, and magnetic polarity, and supports earlier estimates of the age of the sedimentary section; all are beyond 14C range. Four till units are recognized and documented, and 16 subunits are identified. All are ascribed to deposition during a Late Pliocene glaciation that was probably the last time the entire Vestfold Hills was covered by an enlarged East Antarctic Ice Sheet (EAIS). Evidence for other more recent glacial events of the 'Vestfold Glaciation' may have been due to lateral expansion of the Sorsdal Glacier and limited expansion of the icesheet margin during the Last Glacial Maximum rather than a major expansion of the EAIS. The deposit appears to correlate with a marine deposition event recorded in Ocean Drilling Program Site 1166 in Prydz Bay, possibly with the Bardin Bluffs Formation of the Prince Charles Mountains and with part of the time represented in the ANDRILL AND-1B core in the Ross Sea.

d13C and d15N stable isotopes values, total nitrogen and C/N ratios of sediment core GeoB4801-1

Rising stable nitrogen isotope ratios (d15N) in dated sediment records of the German Bight/SE North Sea track river-induced coastal eutrophication over the last 2 centuries. Fully exploiting their potential for reconstructions of pristine conditions and quantitative analysis of historical changes in the nitrogen cycle from these sediment records requires knowledge on processes that alter the isotopic signal in non-living organic matter (OM) of sinking particles and sediments. In this study, we analyze the isotopic composition of particulate nitrogen (PN) in the water column during different seasons, in surface sediments, and in sediment cores to assess diagenetic influences on the isotopic composition of OM. Amino acid (AA) compositions of suspended matter, surface sediments, and dated cores at selected sites of the German Bight serve as indicators for quality and degradation state of PN. The d15N of PN in suspended matter had seasonal variances caused by two main nitrate sources (oceanic and river) and different stages of nitrate availability during phytoplankton assimilation. Elevated d15N values (> 20 per mil) in suspended matter near river mouths and the coast coincide with a coastal water mass receiving nitrate with elevated isotope signal (d15N > 10 per mil) derived from anthropogenic input. Particulate nitrogen at offshore sites fed by oceanic nitrate having a d15N between 5 and 6 per mil had low d15N values (< 2 per mil), indicative of an incipient phytoplankton bloom. Surface sediments along an offshore-onshore transect also reflect the gradient of low d15N of nitrate in offshore sites to high values near river mouths, but the range of values is smaller than between the end members listed above and integrates the annual d15N of detritus. Sediment cores from the coastal sector of the gradient show an increasing d15N trend (increase of 2.5 per mil) over the last 150 years. This is not related to any change in AA composition and thus reflects eutrophication. The d15N signals from before AD 1860 represent a good estimation of pre-industrial isotopic compositions with minimal diagenetic overprinting. Rising d13C in step with rising d15N in these cores is best explained by increasing productivity caused by eutrophication.

(Table 1) Concentrations of amino acids in sediments from DSDP Site 68-502

In this chapter, we will report on the amino acids in the total acid hydrolysate of eight sediment samples from Leg 68 Site 502. This site was located on a topographic high at a depth of 3051 meters in the Colombian Basin of the western Caribbean Sea. Four holes were cored at the site by means of the hydraulic piston corer to a maximum sediment depth of 218 meters. The composite section is a virtually continuous, undisturbed sediment record covering almost 8 million years from the Holocene to late Miocene. Age estimates for the section are based on excellent magnetostratigraphic and biostratigraphic records. Four lithostratigraphic units (A, B, C, and D) were recognized, based on differences in color and content of clay, ash, foraminifers, and siliceous microfossils (Prell, Gardner, et al., 1980): A, yellowish brown to light brownish gray foraminifer-bearing (> 10%) nannofossil marl; B, gray to olive gray foraminifer-bearing nannofossil marl with occasional ash beds; C, light gray to dark greenish gray calcareous clay and foraminifer-bearing (< 10%) nannofossil marl; D, pale green to grayish green calcareous, ash-bearing clay with siliceous microfossils. The calcium carbonate content of these sediments increases from about 27 to about 49% from late Miocene to middle Pliocene (about 3.6 Ma) and remains uniform at about 48 to 50% from that time throughout the Quaternary. The eight sediment samples for amino acid analyses came from the third (502B) and fourth (502C) holes at Site 502. Samples ranged in sub-bottom depth from 4.3 to 225 meters spanning time from 0.3 to 7.7 Ma.

Distribution of major carbon pools in a cyanobacterial mat from Salin de Giraud, Camargue, France

The fine-scale depth distribution of major carbon pools and their stable carbon isotopic signatures (d13C) were determined in a cyanobacterial mat (Salin-de-Giraud, Camargue, France) to study early diagenetic alterations and the carbon preservation potential in hypersaline mat ecosystems. Particular emphasis was placed on the geochemical role of extracellular polymeric substances (EPS). Total carbon (Ctot), organic carbon (Corg), total nitrogen (Ntot), total hydrolysable amino acids (THAA), carbohydrates, cyanobacteria-derived hydrocarbons (8-methylhexadecane, n-heptadec-5-ene, n-heptadecane) and EPS showed highest concentrations in the top millimetre of the mat and decreased with depth. The hydrocarbons attributed to cyanobacteria showed the strongest decrease in concentration with depth. This correlated well with the depth profiles of oxygenic photosynthesis and oxygen, which were detected in the top 0.6 and 1.05 mm, respectively, at a high down-welling irradiance (1441 µmol photons m**-2 s**-1). At depths beneath the surface layer, the Corg was composed mainly of amino acids and carbohydrates. A resistance towards microbial degradation could have resulted from interactions with diverse functional groups present in biopolymers (EPS) and with minerals deposited in the mat. A 13C enrichment with depth for the total carbon pool (Ctot) was observed, with d13C values ranging from -16.3 permil at the surface to -11.3 permil at 9-10 mm depth. Total lipids depicted a d13C value of -17.2 permil in the top millimetre and then became depleted in 13C with depth (-21.7 to -23.3 permil). The d13C value of EPS varied only slightly with depth (-16.1 to -17.3 permil) and closely followed the d13C value of Corg at depths beneath 4 mm. The EPS represents an organic carbon pool of preservation potential during early stages of diagenesis in recent cyanobacterial mats as a result of a variety of possible interactions. Their analyses might improve our understanding of fossilized microbial remains from mat ecosystems.

Messenger ribonucleic acid of cerebral nuclei.

1. RNA was isolated from crude nuclear preparations and from ribosomes derived from rat brain and liver. Nuclear RNA was obtained by lysis of the nuclei with sodium dodecyl sulphate, followed by denaturation and removal of DNA and protein with hot phenol. 2. Base composition analyses indicated that the cerebral nuclear RNA preparation contained a higher proportion of non-ribosomal RNA than the analogous hepatic preparation. 3. Sucrose-density-gradient analyses revealed a heterogeneous profile for each nuclear RNA preparation, with two major peaks possessing the sedimentation properties of ribosomal RNA (18s and 28s). 4. Template activities of both preparations were widely distributed through the sucrose density gradients. 5. The cerebral nuclear RNA preparation was more active than the hepatic nuclear RNA preparation in promoting amino acid incorporation in cell-free systems from Escherichia coli and rat brain. 6. Cerebral nuclear RNA stimulated amino acid incorporation in a cerebral ribosomal system even in the presence of an excess of purified E. coli transfer RNA. 7. It is concluded that a significant proportion of cerebral nuclear RNA has the characteristics of messenger RNA.

Protein quality and distribution determines anabolic signaling, cellular energetics, and body composition

Building and maintaining muscle is critical to the quality of life for adults and elderly. Physical activity and nutrition are important factors for long-term muscle health. In particular, dietary protein – including protein distribution and quality – are under-appreciated determinants of muscle health for adults. The most unequivocal evidence for the benefit of optimal dietary protein at individual meals is derived from studies of weight management. During the catabolic condition of weight loss, higher protein diets attenuate loss of lean tissue and partition weight loss to body fat when compared with commonly recommended high carbohydrate, low protein diets. Muscle protein turnover is a continuous process in which proteins are degraded, and replaced by newly synthesized proteins. Muscle growth occurs when protein synthesis exceeds protein degradation. Regulation of protein synthesis is complex, with multiple signals influencing this process. The mammalian target of rapamycin (mTORC1) pathway has been identified as a particularly important regulator of protein synthesis, via stimulation of translation initiation. Key regulatory points of translation initiation effected by mTORC1 include assembly of the eukaryotic initiation factor 4F (eIF4F) complex and phosphorylation of the 70 kilodalton ribosomal protein S6 kinase (S6K1). Assembly of the eIF4F initiation complex involves phosphorylation of the inhibitory eIF4E binding protein-1 (4E-BP1), which releases the initiation factor eIF4E and allows it to bind with eIF4G. Binding of eIF4E with eIF4G promotes preparation of the mRNA for binding to the 43S pre-initiation complex. Consumption of the amino acid leucine (Leu) is a key factor determining the anabolic response of muscle protein synthesis (MPS) and mTORC1 signaling to a meal. Research from this dissertation demonstrates that the peak activation of MPS following a complete meal is proportional to the Leu content of a meal and its ability to elevate plasma Leu. Leu has also been implicated as an inhibitor of muscle protein degradation (MPD). In particular, there is evidence suggesting that in muscle wasting conditions Leu supplementation attenuates expression of the ubiquitin-proteosome pathway, which is the primary mode of intracellular protein degradation. However, this is untested in healthy, physiological feeding models. Therefore, an experiment was performed to see if feeding isonitrogenous protein sources with different Leu contents to healthy adult rats would differentially impact ubiquitin-proteosome (protein degradation) outcomes; and if these outcomes are related to the meal responses of plasma Leu. Results showed that higher Leu diets were able to attenuate total proteasome content but had no effect on ubiquitin proteins. This research shows that dietary Leu determines postprandial muscle anabolism. In a parallel line of research, the effects of dietary Leu on changes in muscle mass overtime were investigated. Animals consuming higher Leu diets had larger gastrocnemius muscle weights; furthermore, gastrocnemius muscle weights were correlated with postprandial changes in MPS (r=0.471, P<0.01) and plasma Leu (r=0.400, P=0.01). These results show that the effect of Leu on ubiquitin-proteosome pathways is minimal for healthy adult rats consuming adequate diets. Thus, long-term changes in muscle mass observed in adult rats are likely due to the differences in MPS, rather than MPD. Factors determining the duration of Leu-stimulated MPS were further investigated. Despite continued elevations in plasma Leu and associated translation initiation factors (e.g., S6K1 and 4E-BP1), MPS returned to basal levels ~3 hours after a meal. However, administration of additional nutrients in the form of carbohydrate, Leu, or both ~2 hours after a meal was able to extend the elevation of MPS, in a time and dose dependent manner. This effect led to a novel discovery that decreases in translation elongation activity was associated with increases in activity of AMP kinase, a key cellular energy sensor. This research shows that the Leu density of dietary protein determines anabolic signaling, thereby affecting cellular energetics and body composition.

Enzymatic and structural characterization of new PLA2 isoform isolated from white venom of Crotalus durissus ruruima

This work reports the structural and enzymatic characterization of a new sPLA2 from the white venom of Crotalus durissus ruruima, nominated PLA2A. The homogeneity of the PLA2A fraction and its molecular mass were initially evaluated by SDS-PAGE and confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 14,299.34 Da. Structural investigation, through circular dichroism spectroscopy, revealed that PLA2A has a high content of alpha helix and beta-turn structures, 45.7% and 35.6% respectively. Its amino acid sequence, determined by Edman degradation and de novo amino acid sequencing, exhibited high identity to PLA2 Cdt F15 from Crotalus durissus terrificus. The enzymatic investigation, conducted using the synthetic substrate 4-nitre-3-(octanoyloxy)benzoic acid, determined its V(max) (7.56 nmoles/min) and K(M) (2.76 mM).Moreover, PLA2A showed an allosteric behavior and its enzymatic activity was dependent on Ca(2+). Intrinsic fluorescence measurements suggested that Ca(2+) induced a significant increase of PLA2A fluorescence, whereas its replacement for Mg(2+), Mn(2+), Sn(2+) and Cd(2+) apparently induced no structural modifications. The optimal pH and temperature for the enzymatic activity of PLA2A were 8.4 and 40 degrees C, respectively, and the minimal concentration of p-BPB and crotapotin that significantly inhibited such activity was 0.75 mM and 0.4 mu M, respectively. In addition, PLA2A showed a significant antibacterial effect that was not strictly dependent on the enzymatic activity of such sPLA2. (c) 2008 Elsevier Ltd. All rights reserved.

A Catalytically Inactive Lys49 PLA2 Isoform from Bothrops jararacussu venom that Stimulates Insulin Secretion in Pancreatic Beta Cells

A new secretory phospholipase A2 (sPLA2) isoform from Bothrops jararacussu venom (BjVIII) has been characterized by causing platelet aggregation, an absent activity in BthTx-I, Prtx-I and PrTx-II sPLA2s. According to our results, BjVIII also enhances insulin release by the pancreatic beta cells. The complete amino acid sequence of the new isoform was determined by Edman degradation and de novo peptide sequencing. These analyses showed a G35K amino acid modification for BjVIII in comparison with BthTx-I, PrTx-I and Prtx-II, a structural difference that has been related to the conflicting biological activities among BjVIII and other Lys49 sPLA2s. The whole set of evidences collected in this work indicates that, besides the C-terminal region and B-wing of PLA2, the calcium binding loop in BjVIII should be considered as an important region, involved in the pharmacological effects of Lys49-sPLA2 isoforms from the Bothrops genus.

Comprendere come lo stato genetico di TP53 influenza la risposta al deficit indotto sul Complesso I mitocondriale come terapia anticancro

Il Complesso I (CI) mitocondriale è uno dei target metabolici più promettenti nelle terapie anti- cancro. In particolare, la metformina è un inibitore noto del CI, capace di inibire la crescita delle cellule tumorali, ma non di eradicare la patologia. Recentemente, l’associazione metformina ed ipoglicemia si è rivelata letale per i tumori, sebbene l’efficacia terapeutica del trattamento sinergico possa essere influenzata dall’accumulo di alterazioni genetiche nei più noti drivers della tumorigenesi. Abbiamo così investigato l’effetto dello stress metabolico indotto dalla restrizione di glucosio in un pannello di linee cellulari tumorali con un severo deficit sul CI e con un diverso stato genetico di TP53. Il deficit del CI associato alla carenza di glucosio inducono un abbattimento dei livelli di espressione della proteina p53 mutata, ma non della controparte wild-type. Il fenomeno biologico osservato non dipende né da un blocco trascrizionale, né dall’innesco di vie di degradazione intracellulare, come proteasoma ed autofagia. La scomparsa di p53 mutata, invece, sembra dipendere da un blocco generale della sintesi proteica, verosimilmente indotto dallo stress energetico e nutrizionale. Nella controparte p53 wild-type, invece, si osserva solo una parziale riduzione della sintesi proteica, suggerendo l’innesco di possibili vie di adattamento per compensare il danno sul CI. La carenza di amminoacidi è una caratteristica dei tumori solidi che potrebbe essere esacerbata in condizioni di deficit generali della catena respiratoria mitocondriale. In particolare, l’inibizione del CI causa auxotrofia da aspartato, metabolita limitante per la proliferazione, condizione che potrebbe generare il blocco della sintesi proteica osservato. L’incremento di espressione dei livelli del trasportatore aspartato/glutatammato mediata da p53 mutata compensa l’auxotrofia da aspartato, identificando un meccanismo di adattamento al deficit del CI. Dunque, i risultati ottenuti sottolineano l’importanza di implementare la terapia anti-complesso I nel cancro, poiché il diverso stato di p53 può alterare l’efficacia del trattamento.

Molecular And Biochemical Characterization Of Caffeine Synthase And Purine Alkaloid Concentration In Guarana Fruit.

Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein.

Homozygous Inactivating Mutation In Nanos3 In Two Sisters With Primary Ovarian Insufficiency.

Despite the increasing understanding of female reproduction, the molecular diagnosis of primary ovarian insufficiency (POI) is seldom obtained. The RNA-binding protein NANOS3 poses as an interesting candidate gene for POI since members of the Nanos family have an evolutionarily conserved function in germ cell development and maintenance by repressing apoptosis. We performed mutational analysis of NANOS3 in a cohort of 85 Brazilian women with familial or isolated POI, presenting with primary or secondary amenorrhea, and in ethnically-matched control women. A homozygous p.Glu120Lys mutation in NANOS3 was identified in two sisters with primary amenorrhea. The substituted amino acid is located within the second C2HC motif in the conserved zinc finger domain of NANOS3 and in silico molecular modelling suggests destabilization of protein-RNA interaction. In vitro analyses of apoptosis through flow cytometry and confocal microscopy show that NANOS3 capacity to prevent apoptosis was impaired by this mutation. The identification of an inactivating missense mutation in NANOS3 suggests a mechanism for POI involving increased primordial germ cells (PGCs) apoptosis during embryonic cell migration and highlights the importance of NANOS proteins in human ovarian biology.