Two doses of humanized anti-CD25 antibody in renal transplantation: A preliminary comparative study

HuCD25mAb is a humanized anti-CD25 antibody which has the same amino acid sequence as daclizumab (Zenapax, Roche). HuCD25mAb is expressed in Chinese hamster ovary (CHO) cells while daclizumab is expressed in the NSO myeloma cell line. A comparative study was performed to evaluate the pharmacokinetics and pharmacodynamics between huCD25mAb and daclizumab in a two-dose regimen incorporating triple immunosuppressant treatment regimens (MMF, CsA and steroids). Fifteen patients were enrolled and randomized to receive intravenous infusion of either huCD25mAb (n = 10) or daclizumab (n = 5) at a dosage of 1 mg·kg⁻¹ on operation day 0 and post-operation day 14. Serum concentrations of huCD25mAb and daclizumab were measured by a validated competitive ELISA. Subgroups of CD3⁺, CD25⁺, CD4⁺ and CD8⁺ lymphocytes were monitored periodically by flow cytometry. The concentration-time curves of huCD25mAb and daclizumab were found to fit well to a one-compartment model. A significant decline of proportion (%) of CD3-CD25⁺ and CD3⁺CD25⁺ lymphocytes was observed 30 min after first infusion on day 0 (3.40 ± 1.83 to 0.03 ± 0.07, 3.35 ± 2.02 to 0.37 ± 0.49), and these levels remained low for at least 70 days (0.03 ± 0.05, 0.31 ± 0.47). All pharmacokinetic parameters of huCD25mAb seemed similar to those of daclizumab. The two-dose huCD25mAb regimen was as effective as daclizumab in rapidly achieving high therapeutic concentration in the treated patients, and a significant decrease of CD3⁻CD25⁺ and CD3⁺CD25⁺ lymphocytes was demonstrated. This suggests that two-dose regimen is feasible in maintaining host immunosuppression and may provide an effective and economical strategy for reducing incidence of acute graft rejection.Key words: CD25, pharmacokinetics, kidney transplantation, enzyme immunoassay, flow cytometry, monoclonal antibody     

Influence of light on ascorbate formation and metabolism in apple fruits

To further understand the regulatory mechanism of light on the formation of ascorbic acid (AsA) in the sink organs of plants, a systematical investigation on AsA levels, activities of two key biosynthsis enzymes and their mRNA expression as well as the recycling was performed in the fruits of apple (Malus domestica Borkh), under different levels of shade. After the whole trees were shaded with the sun-light about 50–55% for 20 days, AsA levels were significantly decreased in fruit peel, flesh and leaves, while mRNA expression levels and activities of l-galactose dehydrogenase (l-GalDH, EC 1.1.1.117) and l-galactono-1,4-lactone dehydrogenase (l-GalLDH, EC 1.3.2.3) as well as activities of recycling enzymes was clearly declined in the leaf and peel but not in the flesh. By shading fruits only for 20 days, AsA levels, relative mRNA levels and activities of l-GalDH and l-GalLDH as well as activities of recycling enzymes all showed obvious decrease in the peel, but not in the flesh. However, their levels in the peel were markedly increased after the full shade was removed and re-exposed these fruits on natural light for 5 days. It is concluded that light affects AsA biosynthesis and recycling in the peel and leaf, but did not in the fresh. Results also suggest that apple fruit is potential to biosynthesize AsA via the l-galactose pathway, and AsA content in the fruits may depend partly on levels of AsA or other photochemistry controlled by light in the leaves.     

Oligomeric Structure of the Human Reduced Folate Carrier: IDENTIFICATION OF HOMO-OLIGOMERS AND DOMINANT-NEGATIVE EFFECTS ON CARRIER EXPRESSION AND FUNCTION*

The ubiquitously expressed reduced folate carrier (RFC) is the major transport system for folate cofactors in mammalian cells and tissues. Previous considerations of RFC structure and mechanism were based on the notion that RFC monomers were sufficient to mediate transport of folate and antifolate substrates. The present study examines the possibility that human RFC (hRFC) exists as higher order homo-oligomers. By chemical cross-linking, transiently expressed hRFC in hRFC-null HeLa (R5) cells with the homobifunctional cross-linker 1,3-propanediyl bis-methanethiosulfonate and Western blotting, hRFC species with molecular masses of hRFC homo-oligomers were identified. Hemagglutinin- and Myc epitope-tagged hRFC proteins expressed in R5 cells were co-immunoprecipitated from both membrane particulate and surface-enriched membrane fractions, indicating that oligomeric hRFC is expressed at the cell surface. By co-expression of wild type and inactive mutant S138C hRFCs, combined with surface biotinylation and confocal microscopy, a dominant-negative phenotype was demonstrated involving greatly decreased cell surface expression of both mutant and wild type carrier caused by impaired intracellular trafficking. For another hRFC mutant (R373A), expression of oligomeric wild type-mutant hRFC was accompanied by a significant and disproportionate loss of wild type activity unrelated to the level of surface carrier. Collectively, our results demonstrate the existence of hRFC homo-oligomers. They also establish the likely importance of these higher order hRFC structures to intracellular trafficking and carrier function.Folates are members of the B class of vitamins that are required for the synthesis of nucleotide precursors, serine, and methionine in one-carbon transfer reactions (1). Because mammals cannot synthesize folates de novo, cellular uptake of these derivatives is essential for cell growth and tissue regeneration (2, 3). Folates are hydrophilic anionic molecules that do not cross biological membranes by diffusion alone, so it is not surprising that sophisticated membrane transport systems have evolved to facilitate their accumulation by mammalian cells.The ubiquitously expressed reduced folate carrier (RFC)² is widely considered to be the major transport system for folate co-factors in mammalian cells and tissues (3, 4). RFC plays a generalized role in folate transport and provides specialized tissue functions such as transport across the basolateral membrane of renal proximal tubules (5), transplacental transport of folates (6), and folate transport across the blood-brain barrier (7), although the contribution of RFC to intestinal absorption of folates remains controversial (8, 9). Loss of RFC expression or function portends potentially profound physiologic and developmental consequences associated with folate deficiency (10). RFC is also a major transporter of antifolate drugs used for cancer chemotherapy such as methotrexate (Mtx), pemetrexed, and raltitrexed (4). Loss of RFC expression or synthesis of mutant RFC protein in tumor cells results in antifolate resistance caused by incomplete inhibition of cellular enzyme targets and low levels of antifolate substrate for polyglutamate synthesis (4, 11).Reflecting its particular physiologic and pharmacologic importance, interest in RFC structure and function has been high. Since 1994, when murine RFC was first cloned (12), application of state-of-the-art molecular biology and biochemistry methods for characterizing polytopic membrane proteins has led to a progressively detailed picture of the molecular structure of the carrier, including its membrane topology, N-glycosylation, functionally or structurally important domains and amino acids, and packing of α-helix transmembrane domains (TMDs) (4, 13). Although no crystal structure for RFC has yet been reported, a detailed homology model for human RFC (hRFC) based on the bacterial lactose/proton symporter LacY and glycerol 3-phosphate/inorganic phosphate antiporter GlpT was generated (13, 14) that permits testing of hypotheses related to hRFC structure and mechanism in a manner not previously possible.Considerations of hRFC structure and mechanism to date have all been based on the notion that a single 591-amino acid hRFC molecule is sufficient to mediate concentrative uptake of folate and antifolate substrates. However, a growing literature suggests that quaternary structure involving the formation of higher order oligomers (e.g. dimers, tetramers, etc.) is commonly an important feature of the structure and function of many membrane transporters (15-18). For major facilitator superfamily proteins, both monomeric (e.g. LacY, GlpT, UhpT, and GLUT3) (19-22) and oligomeric (e.g. LacS, AE1, GLUT1, and TetA) (23-28) structures have been reported, establishing the lack of a clear structural consensus for these related proteins.In this report, we explore the question of whether hRFC exists as a homo-oligomeric species composed of multiple hRFC monomers. Based on results with an assortment of biochemical methods with wt and a collection of mutant hRFC proteins, we not only demonstrate the existence of oligomeric hRFC but also establish the probable importance of these higher order structures to intracellular trafficking and carrier function.     

观赏桃ISSR-PCR反应体系的优化

采用改良CTAB法从观赏桃满天红叶片中提取基因组DNA,通过单因素实验探讨了模板DNA、Mg~(2+)、dNTPs和Taq DNA酶等条件对观赏桃ISSR-PCR扩增结果的影响,建立了ISSR-PCR扩增的最佳体系:2.5μl反应体系中包含10×Buffer 2.5μl,模板DNA 40ng,Mg~(2+)浓度2.5mmol/L,引物浓度04 μmol/L,dNTPs浓度0.4mmol/L,Taq DNA酶0.5U.利用所建立的体系对红叶桃、菊花桃和春艳等13份材料进行检验,其结果表明优化后的体系适合观赏桃的ISSR-PCR反应.     

Development and Application of a Real-time PCR Method for Pharmacokinetic and Biodistribution Studies of Recombinant Adenovirus

A replication-deficient recombinant adenovirus (Ad5-LFA-3/IgG₁) that encodes secreted LFA-3/IgG₁ was constructed for gene therapy treatment of psoriasis. The purpose of this study was to develop a real-time PCR method for pharmacokinetic and biodistribution studies of Ad5-LFA-3/IgG₁ within the circulation and organs. This method showed good specificity, sensitivity and reproducibility over a wide dynamic range of concentrations. Quantitative measurement of recombinant adenoviral DNA suggested that the level of Ad5-LFA-3/IgG₁ DNA in circulating blood peaked within 10 min following intravenous injection in rhesus macaques. Following this peak, the adenoviral DNA level dropped significantly to a very low level. Real-time PCR revealed that Ad5-LFA-3/IgG₁ DNA was enriched in the spleen, lung and liver after injection of the adenovirus into rats through the tail vein. The adenoviral DNA was barely detected in other tissues. These data provide important information for clinical trials of Ad5-LFA-3/IgG₁ and confirm the utility of the real-time PCR assay for monitoring gene therapy trials.     

Metabolic impact of redox cofactor perturbations in Saccharomyces cerevisiae

Redox cofactors play a pivotal role in coupling catabolism with anabolism and energy generation during metabolism. There exists a delicate balance in the intracellular level of these cofactors to ascertain an optimal metabolic output. Therefore, cofactors are emerging to be attractive targets to induce widespread changes in metabolism. We present a detailed analysis of the impact of perturbations in redox cofactors in the cytosol or mitochondria on glucose and energy metabolism in Saccharomyces cerevisiae to aid metabolic engineering decisions that involve cofactor engineering. We enhanced NADH oxidation by introducing NADH oxidase or alternative oxidase, its ATP-mediated conversion to NADPH using NADH kinase as well as the interconversion of NADH and NADPH independent of ATP by the soluble, non-proton-translocating bacterial transhydrogenase. Decreasing cytosolic NADH level lowered glycerol production, while decreasing mitochondrial NADH lowered ethanol production. However, when these reactions were coupled with NADPH production, the metabolic changes were more moderated. The direct consequence of these perturbations could be seen in the shift of the intracellular concentrations of the cofactors. The changes in product profile and intracellular metabolite levels were closely linked to the ATP requirement for biomass synthesis and the efficiency of oxidative phosphorylation, as estimated from a simple stoichiometric model. The results presented here will provide valuable insights for a quantitative understanding and prediction of cellular response to redox-based perturbations for metabolic engineering applications.     

Caspases regulate VAMP-8 expression and phagocytosis in dendritic cells

During an inflammation and upon encountering pathogens, immature dendritic cells (DC) undergo a maturation process to become highly efficient in presenting antigens. This transition from immature to mature state is accompanied by various physiological, functional and morphological changes including reduction of caspase activity and inhibition of phagocytosis in the mature DC. Caspases are cysteine proteases which play essential roles in apoptosis, necrosis and inflammation. Here, we demonstrate that VAMP-8, (a SNARE protein of the early/late endosomes) which has been shown previously to inhibit phagocytosis in DC, is a substrate of caspases. Furthermore, we identified two putative conserved caspase recognition/cleavage sites on the VAMP-8 protein. Consistent with the up-regulation of VAMP-8 expression upon treatment with caspase inhibitor (CI), immature DC treated with CI exhibits lower phagocytosis activity. Thus, our results highlight the role of caspases in regulating VAMP-8 expression and subsequently phagocytosis during maturation of DC.