植物维生素B_6从头合成与代谢转换研究进展

维生素B6是一组可相互转换的吡啶衍生物的总称,包括吡哆醇、吡哆胺、吡哆醛、磷酸吡哆醇、磷酸吡哆胺和磷酸吡哆醛。其中,磷酸吡哆醛是140多种细胞酶的辅酶。至今发现两种VB6从头合成途径,DXP(1-脱氧-D-木酮糖-5-磷酸)依赖途径和DXP非依赖途径,前者仅存在于大肠杆菌和少量其他细菌,后者存在于其他所有VB6自养生物。除了VB6的从头合成,所有细胞生物体内还存在一条相似的补救途径,补救途径实现VB6各型的代谢转换。该文对近年来国内外有关植物VB6从头合成和代谢转换研究进展进行综述。     

The effects of partial thiamin deficiency and oxidative stress (i.e., glyoxal and methylglyoxal) on the levels of alpha-oxoaldehyde plasma protein adducts in Fischer 344 rats

We hypothesized that in marginal thiamin deficiency intracellular alpha-oxoaldehydes form macromolecular adducts that could possibly be genotoxic in colon cells; and that in the presence of oxidative stress these effects are augmented because of decreased detoxification of these aldehydes. We have demonstrated that reduced dietary thiamin in F344 rats decreased transketolase activity and increased alpha-oxoaldehyde adduct levels. The methylglyoxal protein adduct level was not affected by oral glyoxal or methylglyoxal in the animals receiving thiamin at the control levels but was markedly increased in the animals on a thiamin-reduced diet. These observations are consistent with our suggestion that the induction of aberrant crypt foci with marginally thiamin-deficient diets may be a consequence of the formation of methylglyoxal adducts.     

Inhibition of thymidylate synthase by hydroxyurea in rapidly proliferating P815 mastocytoma cells

The incorporation of [¹⁴C]deoxycytidine, [³H]deoxyuridine, and [³H]thymidine, respectively into pyrimidine bases of DNA has been measured in rapidly proliferating P815 mouse mastocytoma cells in the presence of hydroxyurea. The incorporation of [¹⁴C]deoxycytidine-derived radioactivity into DNA cytosines is increased when compared to the incorporation into DNA thymines. The [³H]deoxyuridine-derived radioactivity is incorporated solely into DNA thymines and this incorporation is inhibited by hydroxyurea in a dose-dependent manner. This suggests an inhibitory effect of hydroxyurea on the thymidylate synthase which was proved in experiments in which the conversion of deoxyuridine monophosphate into deoxythymidine monophosphate catalysed by a crude enzyme preparation from P815 cells was inhibited in the presence of hydroxyurea. Enzymatic DNA methylation as measured by the conversion of incorporated [¹⁴C]deoxycytidine into 5-methylcytosines was not affected by hydroxyurea.     

Seven day oral supplementation with Cardax TM (disodium disuccinate astaxanthin) provides significant cardioprotection and reduces oxidative stress in rats

In the current study, the improved oral bioavailability of a synthetic astaxanthin derivative (Cardax^TM; disodium disuccinate astaxanthin) was utilized to evaluate its potential effects as a cardioprotective agent after 7-day subchronic oral administration as a feed supplement to Sprague-Dawley rats. Animals received one of two concentrations of Cardax^TM in feed (0.1 and 0.4%; ∼125 and 500 mg/kg/day, respectively) or control feed without drug for 7 days prior to the infarct study carried out on day 8. Thirty minutes of occlusion of the left anterior descending (LAD) coronary artery was followed by 2 h of reperfusion prior to sacrifice, a regimen which resulted in a mean infarct size (IS) as a percentage (%) of the area at risk (AAR; IS/AAR,%) of 61 ± 1.8%. The AAR was quantified by Patent blue dye injection, and IS was determined by triphenyltetrazolium chloride (TTC) staining. Cardax^TM at 0.1 and 0.4% in feed for 7 days resulted in a significant mean reduction in IS/AAR,% to 45 ± 2.0% (26% salvage) and 39 ± 1.5% (36% salvage), respectively. Myocardial levels of free astaxanthin achieved after 7-day supplementation at each of the two concentrations (400 ± 65 nM and 1634 ± 90 nM, respectively) demonstrated excellent solid-tissue target organ loading after oral supplementation. Parallel trends in reduction of plasma levels of multiple lipid peroxidation products with disodium disuccinate astaxanthin supplementation were observed, consistent with the documented in vitro antioxidant mechanism of action. These results extend the potential utility of this compound for cardioprotection to the elective human cardiovascular patient population, for which 7-day oral pre-treatment (as with statins) provides significant reductions in induced periprocedural infarct size.     

Identification of the putative tumor suppressor Nit2 as ω-amidase, an enzyme metabolically linked to glutamine and asparagine transamination

The present report identifies the enzymatic substrates of a member of the mammalian nitrilase-like (Nit) family. Nit2, which is widely distributed in nature, has been suggested to be a tumor suppressor protein. The protein was assumed to be an amidase based on sequence homology to other amidases and on the presence of a putative amidase-like active site. This assumption was recently confirmed by the publication of the crystal structure of mouse Nit2. However, the in vivo substrates were not previously identified. Here we report that rat liver Nit2 is ω-amidodicarboxylate amidohydrolase (E.C. 3.5.1.3; abbreviated ω-amidase), a ubiquitously expressed enzyme that catalyzes a variety of amidase, transamidase, esterase and transesterification reactions. The in vivo amidase substrates are α-ketoglutaramate and α-ketosuccinamate, generated by transamination of glutamine and asparagine, respectively. Glutamine transaminases serve to salvage a number of α-keto acids generated through non-specific transamination reactions (particularly those of the essential amino acids). Asparagine transamination appears to be useful in mitochondrial metabolism and in photorespiration. Glutamine transaminases play a particularly important role in transaminating α-keto-γ-methiolbutyrate, a key component of the methionine salvage pathway. Some evidence suggests that excess α-ketoglutaramate may be neurotoxic. Moreover, α-ketosuccinamate is unstable and is readily converted to a number of hetero-aromatic compounds that may be toxic. Thus, an important role of ω-amidase is to remove potentially toxic intermediates by converting α-ketoglutaramate and α-ketosuccinamate to biologically useful α-ketoglutarate and oxaloacetate, respectively. Despite its importance in nitrogen and sulfur metabolism, the biochemical significance of ω-amidase has been largely overlooked. Our report may provide clues regarding the nature of the biological amidase substrate(s) of Nit1 (another member of the Nit family), which is a well-established tumor suppressor protein), and emphasizes a) the crucial role of Nit2 in nitrogen and sulfur metabolism, and b) the possible link of Nit2 to cancer biology.     

Synthesis of branched 9-[2-(2-phosphonoethoxy)ethyl]purines as a new class of acyclic nucleoside phosphonates which inhibit Plasmodium falciparum hypoxanthine–guanine–xanthine phosphoribosyltransferase

The malarial parasite Plasmodium falciparum (Pf) lacks the de novo pathway and relies on the salvage enzyme, hypoxanthine–guanine–xanthine phosphoribosyltransferase (HGXPRT), for the synthesis of the 6-oxopurine nucleoside monophosphates. Specific acyclic nucleoside phosphonates (ANPs) inhibit PfHGXPRT and possess anti-plasmodial activity. Two series of novel branched ANPs derived from 9-[2-(2-phosphonoethoxy)ethyl]purines were synthesized to investigate their inhibition of PfHGXPRT and human HGPRT. The best inhibitor of PfHGXPRT has a K_i of 1 μM. The data showed that both the position and nature of the hydrophobic substituent change the potency and selectivity of the ANPs.     

RNA turnover and protein synthesis in fish cells

Protein synthesis in fish has been previously correlated with RNA content. The present study investigates whether protein and RNA synthesis rates are similarly related. Protein and RNA synthesis rates were determined from ³H-phenylalanine and ³H-uridine incorporation, respectively, and expressed as % · day⁻¹ and half-lives, respectively. Three fibroblast cell lines were used: BF-2, RTP, CHSE 214, which are derived from the bluegill, rainbow trout and Chinook salmon, respectively. These cells contained similar RNA concentrations (∼175 μg RNA · mg⁻¹ cell protein). Therefore differences in protein synthesis rates, BF-2 (31.3 ± 1.8)>RTP (25.1 ± 1.7)>CHSE 214 (17.6 ± 1.1), were attributable to RNA translational efficiency. The most translationally efficient RNA (BF-2 cells), 1.8 mg protein synthesised · μg⁻¹ RNA · day⁻¹, corresponded to the lowest RNA half-life, 75.4 ± 6.4 h. Translationally efficient RNA was also energetically efficient with BF-2 cells exploiting the least costly route of nucleotide supply (i.e. exogenous salvage) 3.5–6.0 times more than the least translationally efficient RNA (CHSE 214 cells). These data suggest that differential nucleotide supply, between intracellular synthesis and exogenous salvage, constitutes the area of pre-translational flexibility exploited to maintain RNA synthesis as a fixed energetic cost component of protein synthesis. Accepted: 12 November 1999     

Economic Analysis of the Legal Regimes Governing Salvage of Historic Shipwrecks

This article offers an economic analysis of the international and U.S. laws governing the recovery of archaeological data from historic shipwrecks. The framework combines values of treasure salvage and archaeological knowledge. It is suggested that U.S. salvage law, sometimes extended to international waters, gives insufficient protection to archaeological value, but that UNESCO's Convention on the Protection of the Underwater Cultural Heritage goes too far in the other direction. Two other legal regimes (government-salvager and interstate agreements) are shown to have the potential to maximize the social values of historic shipwrecks. It is also suggested that a move towards maximizing social values would be promoted if the U.S. admiralty courts tied the size of salvage awards more closely to the quality of the archaeological work performed.     

Trypanosoma brucei: a survey of pyrimidine transport activities

Purine uptake has been studied in many protozoan parasites in the last few years, and several of the purine transporters have been cloned. In contrast, very little is known about the salvage of preformed pyrimidines by protozoa, and no pyrimidine transporters have been cloned, yet chemotherapy based on pyrimidine nucleobases and nucleosides has been as effective as purine antimetabolites in the treatment of infectious and neoplastic disease. Here, we surveyed the presence of pyrimidine transporters in Trypanosoma brucei brucei. We could not detect any mediated uptake of thymine, thymidine or cytidine, but identified a very high-affinity transporter for cytosine, designated C1, with a K(m) value of 0.048+/-0.009 microM. We also confirmed the presence of the previously reported U1 uracil transporter and found it capable of mediating uridine uptake as well, with a K(m) of 33+/-5 microM. A higher-affinity U2 uridine transporter (K(m)=4.1+/-2.1 microM) was also identified, but efficiency of the C1 and U2-mediated transport was low. Pyrimidine antimetabolites were tested as potential trypanocidal agents and only 5-fluorouracil was found to be effective. This drug was efficiently taken up by bloodstream forms of T. b. brucei.     

A nonspecific nucleoside hydrolase from Leishmania donovani: implications for purine salvage by the parasite.

In contrast to their mammalian hosts, protozoan parasites do not synthesize purines de novo, but depend on preformed nucleotides that they purportedly obtain by salvage pathways. Nucleoside hydrolases may play a crucial role in that salvage process. By screening Leishmania donovani libraries with polyclonal antibodies against promastigote soluble exo-antigens, we have identified a cDNA encoding a protein with significant homology to nonspecific and uridine–inosine-preferring nucleoside hydrolases. Sequence comparison demonstrated that all the residues involved in Ca²⁺-binding and substrate recognition in the active site are conserved among the characterized protozoan nucleoside hydrolases. Genomic analysis suggests that it is a single copy gene in L. donovani, and its homologues are present in members representing other Leishmania species complexes. Both Northern blot and immunoblot analyses indicate that it is constitutively expressed in L. donovani promastigotes. The recombinant enzyme overexpressed in and purified from bacteria showed significant activity with all naturally occurring purine and pyrimidine nucleosides, and efficient utilization of p-nitrophenyl-β- -ribofuranoside as a substrate. Altogether, the sequence comparison and substrate specificity data identify this L. donovani nucleoside hydrolase as a nonspecific nucleoside hydrolase. Further, the nucleoside hydrolase was localized to specific foci in L. donovani promastigotes by immunofluorescent assays. Although the conservation of the nucleoside hydrolases among protozoan parasites offers promise for the design of broad-spectrum anti-parasitic drugs, the existence of multiple and distinct nucleoside hydrolases in a single species demands special consideration.