Cobalamin must be metabolized intracellularly in order to bind two enzymes: methionine synthase in cytoplasm and methylmalonyl-CoA mutase in mitochondria. Defects in this process cause different inborn errors of cobalamin metabolism (cblA-cblG and mut). A previous study described a cobalamin-binding protein, in addition to methylmalonyl-CoA mutase, in crude mitochondrial fractions. The amount of [57Co]cobalamin bound to this protein was increased in cblB, mut and cblD variant2 cell lines, compared to control cell lines. In the present study, this protein was identified as transcobalamin (TC). Mitochondrial fractions from a cblB cell line were incubated with anti-TC antibodies, which precipitated the cobalamin-bound protein. Analysis of mitochondrial and cytoplasmic fractions isolated from a chloroquine-incubated cblF cell line showed that isolated mitochondrial fractions contain lysosomal material, suggesting that the identified TC is lysosomal. Quantification of cobalamin-bound TC levels in whole cell extracts showed significant increases in cblB and mut groups compared to control cell lines.

The potential effects of nutritional support with amino acids or dextrose and epidural blockade on the catabolic response to surgery were investigated in diabetic patients undergoing colorectal surgery. Protein and glucose metabolism were assessed with a stable isotope infusion technique using the two stable isotopes L-[1-13C]leucine and [6,6-2H2 ]glucose. / 1. The first intervention of a postoperative infusion of amino acids avoided pronounced hyperglycaemia in diabetic patients after colorectal surgery and achieved a positive protein balance compared to dextrose. / 2. The second intervention of a short term infusion of amino acids postoperatively blunted protein breakdown and stimulated protein synthesis. This resulted in a positive protein balance in patients with epidural blockade compared to patient controlled analgesia with intravenous morphine. With regard to glucose metabolism, amino acid supply after surgery decreased glucose clearance and endogenous glucose production independent from type of analgesia.

Protein wasting is a complication of glucocorticoid (GC) therapy. It causes substantial morbidity and there is no treatment. This thesis investigates the metabolic mechanisms underlying GC-induced protein wasting and the potential for anabolic hormones to reverse protein loss. The models of GC excess were Cushing's syndrome and GC therapy. Whole body protein metabolism was assessed using the leucine turnover technique and body composition by dual-energy X-ray absorptiometry to estimate lean body mass (LBM) and fat mass (FM). As previous studies demonstrated that LBM and FM influenced rates of protein metabolism, the magnitude of body compositional abnormality in Cushing's syndrome was determined. After accounting for the greater FM (30%) and lesser LBM (15%), protein metabolism in Cushing's syndrome was characterised by a significant increase in protein oxidation, an abnormality that leads to irreversible protein loss. Successful treatment of Cushing's syndrome normalised protein oxidation. Studies of the acute and chronic effects of therapeutic GCs revealed a time-dependent effect on protein metabolism. GCs acutely increased protein oxidation. However, the rate of protein oxidation during chronic therapy at a similar dose was not significantly different to untreated control subjects. This time-dependent change suggests that GC-induced stimulation of protein oxidation does not persist and could represent a metabolic adaptation to limit protein loss. This finding contrasts with that in Cushing's syndrome, where protein oxidation is persistently elevated. This difference may represent a dose effect. Studies in GH-deficient subjects revealed that GH induced a fall in protein oxidation that was significantly correlated with a subsequent gain in LBM. This suggests that the anabolic potential of a therapeutic substance can be predicted by its ability to suppress protein oxidation acutely. Finally, the potential for GH and androgens to reverse the metabolic effects of GCs was assessed. A preliminary study in GC users revealed that a GH dose of 0.8 mg/d was effective in reducing protein oxidation. In a subsequent study, the GH-induced reduction in protein oxidation in women on GCs was enhanced by combined treatment with dehydroepiandrosterone, an androgen. In summary, GCs induce protein loss by stimulating protein oxidation. GH reverses this effect and this action is enhanced by coadministration of androgens. GH and androgens may be used therapeutically to prevent protein loss induced by GCs.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2008. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Feb. 13, 2009). Includes bibliographical references (p. 73-93).

Thesis (MSc Food Sc )--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Fermented fish products are popular food products mainly consumed and produced in Southeast Asia. These products are not produced in South Africa, and those available to the public are imported. The main action during the production and fermentation of this sort of product, is that of proteolysis, either by the bacteria or enzymes naturally present in the fish. The prevalent microbes present in six fermented fish samples from Bangkok (Thailand) and seven from Khon Kaen (Thailand), were determined, and using numerical methods, clustered into similarity groups using the calculated dendrogram .distance (Do) technique to determine their relation to reference strains. Forty-seven different bacterial strains were isolated, but no yeasts, moulds or lactic acid bacteria were found. Five Gram-negative, oxidase-positive species, five different Staphylococcus species and nine different endospore-forming species of the genus Bacillus, were isolated and identified using the API systems. The data indicated that members of the genus Bacillus were the prevalent organisms in all the products examined. The isolates were also scanned for general enzyme activity using the API Zym technology, and the production of proteases was investigated using the Standard Methods Caseinate and the Universal Protease Substrate methods. It was found that most of the isolated organisms produced protease, which is of major importance in the production of fermented fish products. Proteolytic cultures from the fermented fish products, as well as lactic acid starters, were used in the production of a fermented fresh water fish product. Production parameters including: glucose, inoculum, moisture content and incubation time, were evaluated in order to select optimum fermentation conditions. Fermentation efficiency was determined by measuring the final pH, titratable acid and the free amino nitrogen content. Optimum efficiency was obtained with 5% (w/w) added glucose at a moisture level of 150 ml water per 100 g fish. A factorial design (3 x 3 x 3) was used to indicate viable trends to facilitate optimisation of the fermentation process. The main effects, two-factor and three-factor interactions were calculated. Optimum trends obtained were a glucose concentration of 5% (wlw) , inoculum concentration of 1x10⁸ kve.ml ̄ ¹, an incubation period of 15 days and temperature of 30°C. Three lactic acid starters (226 - Lactobacillus plantarum, 140 - Lactococcus diacetylactis and 407 - Pediococcus cerevisiae) were selected as they produced some of the best fermentation results and are safe to use in food. It was found that a combination of all three strains (226, 140 and 407) yielded the best results. By using the above parameters, an acceptable product was produced in terms of consistency, colour and aroma. Further studies need to be conducted to optimise the safety of the product as well as the flavour, both chemically and sensorically optimisation of the product. / AFRIKAANSE OPSOMMING: Gefermenteerde visprodukte is populere voedselprodukte in die lande van Suidoos-Asie. Die produkte word nie in Suid-Afrika geproduseer nie, maar slegs ingevoer. Die hoof aksie tydens die fermentasie proses is proteolise deur die bakteriee en ensieme wat natuurlik teenwoordig is in vis. Die oorheersende mikrobes teenwoordig in ses gefermenteerde vis produkte van Bangkok (Thailand) en sewe van Khon Kaen (Thailand), is bepaal. Numeriese metodes is gevolg om die isolate in groepe te sorteer en te groepeer deur gebruik te maak van die berekende dendrogram afstand (Do) tegniek om hul verwantskap ten opsigte van die verwysingsorganisme te bepaal. Sewe-en-veertig verskillende bakteriee is ge·isoleer, maar geen fungi of melksuurbakteriee is ge·identifiseer nie. Vyf Gram-negatiewe, oksidase-positiewe spesies, vyf verskillende Staphylococcus spesies en nege verskillende endospoorvormende spesies van die genus Bacillus, is geisoleer en ge·identifiseer deur gebruik te maak van die API CHB sisteme. Die data het getoon dat lede van die genus Bacillus die oorheersende organismes was. Die isolate is daarna ondersoek vir algehele ensiemaktiwiteit deur van die API Zym tegnologie gebruik te maak. Daar is veral klem gelê op die protease aktiwiteit en dit is gemeet deur van die "Standard Methods Caseinate Agar" metode asook die "Universal Protease Substrate" metodes gebruik te maak. Daar is gevind dat die oorgrote meerderheid organismes proteolitiese ensieme produseer wat belangrik is in die produksie van gefermenteerde visprodukte. Kulture wat ge·isoleer is uit gefermenteerde visprodukte asook melkssuurkulture is gebruik vir die produksie van 'n gefermenteerde varswater visproduk. Produksieparameters insluitende: glukose-, inokulum- en voginhoud asook inkubasie tyd is ondersoek om die optimum fermentasie kondisies te bepaal. Optimum effektiwiteit is gevind by 'n 5% glukose konsentrasie en vogvlakke van 150 ml water per 100 9 vis. 'n Faktoriale ontwerp (3 x 3 x 3) is gebruik om die optimum kondisies te bepaal. Die hoof effekte asook die twee faktor en drie faktor interaksies is bereken. Optimum neigings is gevind by 'n glukose konsentrasie van 5%, inokulum konsentrasie van 1x10⁸ kve.ml ̄ ¹, 'n inkubasie tydperk van 15 dae en temperatuur van 30°C. Drie melksuurbakteriee (226 - Lactobacillus plantarum, 140 - Lactococcus diacetylactis en 407 - Pediococcus cerevisiae) is gekies aangesien hulle die beste resultate gelewer het en veilig vir gebruik in voedselprodukte is. Daar is gevind dat die drie melksuurkulture saam in kombinasie die beste fermentasie resultate opgelewer het. Deur gebruik te maak van die bogenoemde fermentasie kondisies, kon 'n aanvaarbare produk, in terme van kleur en geur, gelewer word. Verdere studies moet gedoen word om die veiligheid asook die geur, chemies asook sensories, te optimiseer.

Bibliography: pages 147-169. / Butyrivibrio fibrisolvens H17c is a gram-negative obligate anaerobic bacterium found in the rumen of most ruminants. The aim of this thesis was to investigate the enzymes produced by B. fibrisolvens H17c involved in the degradation of cellulose, xylan, and protein. A library of chromosomal DNA fragments from B. fibrisolvens H17c was established in the plasmid pEcoR251, an Escherichia coli positive selection vector. The library was screened for genes expressing cellulase, xylanase, and protease activity. Two genes expressing endo-β-1,4-glucanase and cellodextrinase activity were cloned in E. coli as host. The gene expressing endo-β-1,4-glucanase activity (end1) was cloned on a recombinant plasmid pES400. The end1 gene was located on a 6.8 kb DNA fragment and expressed from its own promoter in the E. coli host. It was shown that 64% of the endoglucanase activity was located in the periplasm of the E. coli host. TnphoA mutagenesis indicated the presence of a functional E. coli-like signal peptide. The nucleotide sequence of end1 was determined and the amino acid sequence (547 amino acids) deduced. The catalytic domain of End1 showed very good similarity to the catalytic domain of the Clostridium thermoceiium EGE endoglucanase. End1 also has a non-catalytic domain similar to the binding domains of the CenA and Cex cellulases from Ceilulomonas fimi The gene expressing cellodextrinase activity (ced1) was cloned on a recombinant plasmid pES500. This gene was located on a 3.55 kb fragment and was also expressed from its own promoter in the E. coli host. The Ced1 enzyme was also exported to the periplasm of the E. coli host, but did not contain a functional E. coli-like signal peptide. The nucleotide sequence was determined and the deduced amino acid sequence (547 residues) showed high similarity to the catalytic domain of the C. thermocellum EGD endoglucanase. The proteins of End1 and Ced1 showed no similarity. The End1 and Ced1 enzymes were characterized using a range of different substrates. The End1 enzyme showed optimal activity at pH 5.6 and 45°C. Optimal activity for the Ced1 enzyme was obtained at pH 6.6 and 50°C. The proteolytic activity of B. fibrisolvens H17c was characterized using gelatin-SD5-PAGE. Ten bands of protease activity with apparent molecular weights ranging between 42 000 and 101 000 were detected at different stages during the growth cycle. The effect of protease inhibitors indicated that all ten protease bands were serine proteases. Optimal activity was observed between pH 6.0 to 7.5 and at a temperature of 50°C. The proteolytic activity of B. fibrisolvens H17c varied depending on the type of carbohydrate substrate in the medium, and was positively correlated with the growth rate.

Modification of proteins by ADP-ribose has been shown to be a versatile modification with respect to the amino acid side chain. The results described here will allow the study of the biological importance of ADP-ribose glycation and also allow differentiation on crude extracts between enzymatic modifications from protein ADP-ribose glycation that can occur due to the presence of NAD glycohydrolases.

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