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- Jia, Jia
(författare)
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Crystallographic studies of transaldolase : implications for the enzymatic mechanism and the evolution of class I aldolases
- 1997
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Transaldolase catalyzes the reversible transfer of a dihydroxyacetone moiety from a ketose donor to an aldose acceptor by forming a Schiff-base intermediate between a Iysine residue and dihydroxyacetone. It belongs to the class I aldolase family. A common mechanistic feature of the members of this enzyme family is the formation of a covalent intermediate between an active site Iysine residue and the substrate during catalysis. The three-dimensional structure of recombinant transaldolase B from E.coli has been determined at 1.87 A resolution using the multiple isomorphous replacement method. The current model comprises residues 2-317 and has been refined to an R-factor of 20.1% and R free of 23.4%. The overall structure consists of a single domain, ana/B barrel. The active site is located at the C-terminal end of the B strands. The fold of transaldolase is similar to other enzyme structures in the class I aldolase family. Comparison of these structures suggests that a circular permutation has occurred in the ancestral aldolase gene. This observation provides the first structural evidence for a naturally occurring circular permutation in an a/B barrel protein. The structure of a trapped Schiff-base intermediate complex of this enzyme has been determined at 2.2 A resolution and refined to R-factor of 20.4% and R-free of 24.4%. Tbe structure of the complex provides direct crystallographic evidence for the formation of a Schiff base intermediate in this enzyme family. Based on the structure, a reaction mechanism for transaldolase is proposed. The main features of this mechanism are: Lys 132 acts as the Schiff base forming residue, and two acidic groups, Glu96 and Aspl7 and a catalytic water molecule are involved in proton transfer during the reaction. Several functionally important residues at the active site and dimer interface have been mutated by site-directed mutagenesis and the mutants have been analyzed by crystallography. The structural analysis confirmed the mutations and showed that no unintended structural changes were introduced. The kinetic properties of these mutant enzymes are consistent with the proposed roles for these residues in catalysis.
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| 2. |
- Zhang, Zhi-Jia, et al.
(författare)
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Synergistic inhibitory effects of interferon-alpha and 5-fluorouracil on human meningeoma cells in vitro
- 1996
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Ingår i: Cancer Letters. - : Elsevier BV. - 0304-3835 .- 1872-7980. ; 100:1-2, s. 99-105
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Tidskriftsartikel (refereegranskat)abstract
- We have investigated the effects of interferon-α (IFN-α) and 5-fluorouracil (5-FU) on meningioma cells in two different culture systems, evaluated by the uptake of radiolabelled methionine. With both IFN-α and 5-FU an inhibitory effect on the uptake of radiolabelled methionine by the meningioma cells was demonstrated, and we found a synergistic inhibitory effect with a combination of IFN-α and 5-FU. To obtain a maximal inhibition of cell metabolism without causing cell toxicity, we were able to decrease the dose of 5-FU by simultaneously adding IFN-α. Our results suggest that a combined treatment of IFN-α and 5-FU may be a successful alternative for patients with inoperable meningiomas. A novel in vitro positron emission tomography technique was used for the study of metabolic changes in tumour cells caused by drug treatment, which is complementary to conventional cell culture techniques.
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| 3. |
- Berts, Alf, et al.
(författare)
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Oscillatory Ca2+ signaling in somatostatin-producing cells from the human pancreas
- 1997
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Ingår i: Metabolism. - 0026-0495 .- 1532-8600. ; 46:4, s. 366-369
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Tidskriftsartikel (refereegranskat)abstract
- Oscillatory Ca2+ signaling was studied in human somatostatin-releasing pancreatic δ cells identified by immunostaining. A ratiometric fura-2 technique was used for measuring cytoplasmic concentrations of Ca2+ and Sr2+ in δ cells exposed to the respective cation. Rhythmic activity in terms of slow (frequency, 0.1 to 0.4 per minute) oscillations from close to the basal level was seen in the presence of 3 to 20 mmol/L glucose during superfusion with medium containing 2.6 to 5 mmol/L Ca2+ or 5 mmol/L Sr2. These oscillations could be transformed into a sustained increase by decreasing extracellular Ca2+ or adding 1 mmol/L tolbutamide or 20 nmol/L glucagon. Addition of glucagon to a medium containing 20 mmol/L glucose resulted in the generation of short (< 30 seconds) transients, which disappeared upon exposure to 100 nmol/L of the intracellular Ca2+-adenosine triphosphatase (ATPase) inhibitor thapsigargin. When analyzing small aggregates of islet cells, it became evident that oscillatory activity in δ cells can be synchronous with that in adjacent non—δ cells. It is concluded that secretion of pancreatic somatostatin in man involves Ca2+ signaling similar to that regulating the pulsatile release of insulin.
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| 4. |
- Fang, Jia-Long
(författare)
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Analysis of DNA adducts of some low molecular weight aldehydes : methods development and application in human biomonitoring
- 1997
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Malondialdehyde (MA), acetaldehyde (Aa) and methylglyoxal (MG) are ubiquitously present in the environment and endogenously formed in animals and humans. They have been shown to be genotoxic and to readily react with DNA to form DNA adducts under physiological conditions. The present studies were aimed at developing specific 32p-postlabelling methods for the analysis of these DNA adducts and to apply them to measure these DNA adducts in vitro (MG, Aa) and in vivo in mice (Aa) and in humans (MA, Aa). Endogenously formed DNA adducts of MA were detected in DNA isolated from total white blood cells and from breast tissue of known unexposed healthy individuals by using the 32P-postlabelling technique. A large interindividual variation in adduct levels was observed. The effects of dietary fatty acid composition on the endogenous formation of DNA adducts of MA were investigated in humans ingesting carefully controlled diets. MA-DNA adduct levels in total white blood cell DNA from subjects given a sunflower oil-based (rich in polyunsaturated fatty acids) diet, which resulted in higher concentrations of polyunsaturated fatty acids in plasma triglycerides, were 3.6-fold higher than the corresponding levels in individuals given a rapeseed oil-based (rich in monounsaturated fatty acids) diet. Aa was shown to readily react with deoxynucleosides in a buffered solution at room temperature and neutral pH. AU the adducts formed were chemically unstable at room temperature and neutral pH. Chemically unstable adducts were also formed by the co- operative reaction of Aa with ethanol. Five stable adducts were obtained in the reaction of Aa with deoxyguanosine (dG) after reduction with NaBH4 and structurally characterized. A 32P-postlabelling assay was developed for the determination of N2-ethyl-3'-dGMP, the major stable adduct obtained in vitro, and was shown to be sensitive enough for the detection of adducts in both calf thymus DNA exposed to Aa in vitro and in liver DNA from mice treated with ethanol. Further, the effect of alcohol drinking on the formation of DNA adducts of Aa was investigated in humans. A large interindividual variation in adduct levels was observed. The average adduct levels in granulocyte and Iymphocyte DNA from alcoholic patients were 13- and 7-fold higher than the corresponding levels in the control subjects. Four stable isomeric reaction products formed between MG and dG or 3'-dGMP were separated by reversed-phase HPLC and structurally characterized. A 32P-postlabelling assay was developed for the determination of MG-3'-dGMP adducts and was shown to be sensitive enough for the detection of adducts in both calf thymus DNA and human Iymphocytes after in vitro exposure to MG. The results presented here suggest that DNA adducts of MA and Aa could serve as important biomarkers to assess the contribution of dietary and life-style factors such as fat intake and alcohol drinking, respectively, to human carcinogenesis.
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