Molecular Cloning, Expression, and Pharmacological Characterization of humEAA1, a Human Kainate Receptor Subunit

Abstract: Kainate is a potent neuroexcitatory agent; its neurotoxicity is thought to be mediated by an ionotropic receptor with a nanomolar affinity for kainate. In this report, we describe the cloning of a cDNA encoding a human glutamate ionotropic receptor subunit protein from a human hippocampal library. This cDNA, termed humEAA1, is most closely related to rat and human cDNAs for kainate receptor proteins and, when expressed in COS or Chinese hamster ovary cells, is associated with high-affinity kainate receptor binding. We have successfully established cell lines stably expressing humEAA1. This is the first report of establishment of stable cell lines expressing a glutamate receptor subunit. The relative potency of compounds for displacing [³H] kainate binding of humEAA1 receptors expressed in these stable cell lines was kainate > quisqualate > domoate > L-glutamate > ( RS )-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid > dihydro-kainate > 6, 7-dinitroquinoxaline-2, 3-dione > 6-cyano-7-nitroquinoxaline-2, 3-dione. Homooligomeric expression of humEAA1 does not appear to elicit ligand-gated ion channel activity. Nevertheless, the molecular structure and pharmacological characterization of high-affinity kainate binding of the humEAA1 expressed in the stable cell line (ppEAA1–16) suggest that the humEAA1 is a subunit protein of a human kainate receptor complex.     

Subunit interface mutants of rabbit muscle aldolase form active dimers.

We report the construction of subunit interface mutants of rabbit muscle aldolase A with altered quaternary structure. A mutation has been described that causes nonspherocytic hemolytic anemia and produces a thermolabile aldolase (Kishi H et al., 1987, Proc Natl Acad Sci USA 84:8623-8627). The disease arises from substitution of Gly for Asp-128, a residue at the subunit interface of human aldolase A. To elucidate the role of this residue in the highly homologous rabbit aldolase A, site-directed mutagenesis is used to replace Asp-128 with Gly, Ala, Asn, Gln, or Val. Rabbit aldolase D128G purified from Escherichia coli is found to be similar to human D128G by kinetic analysis, CD, and thermal inactivation assays. All of the mutant rabbit aldolases are similar to the wild-type rabbit enzyme in secondary structure and kinetic properties. In contrast, whereas the wild-type enzyme is a tetramer, chemical crosslinking and gel filtration indicate that a new dimeric species exists for the mutants. In sedimentation velocity experiments, the mutant enzymes as mixtures of dimer and tetramer at 4 degrees C. Sedimentation at 20 degrees C shows that the mutant enzymes are > 99.5% dimeric and, in the presence of substrate, that the dimeric species is active. Differential scanning calorimetry demonstrates that Tm values of the mutant enzymes are decreased by 12 degrees C compared to wild-type enzyme. The results indicate that Asp-128 is important for interface stability and suggest that 1 role of the quaternary structure of aldolase is to provide thermostability.     

Overview of a quality assurance/quality control compliance program consistent with FDA regulations and policies for somatic cell and gene therapies: a four year experience

Somatic cell and gene therapy involve the application of biological technologies to an individual patient through the use of living cells which provide a therapeutic benefit (Aliski, 1991). Various forms of cellular and gene therapies are being developed and evaluated in an increasing number of clinical trials for congential and acquired disorders. The potential and progress of these therapeutic applications have resulted in an increasing effort by the Food and Drug Administration (FDA) to develop the regulatory framework under which these therapeutic approaches would insure safety and efficacy, the primary mandate of the FDA.Over five years ago Cellcor began to define the parameters, specifications, and conditions relevant to a Quality Assurance/Quality Control (QA/QC) program that has evolved to insure safety and maximize the efficacy of applications of the company'sex vivo technology, autolymphocyte therapy. Autolymphocyte therapy is an outpatient form of somatic cell immunotherapy based upon the infusion of T cells that have been activatedex vivo using a combination of previously generated autologous cytokines and an anti-CD3 monoclonal antibody.We have been able to demonstrate the feasibility for the safe, controlled, and consistent preparation and delivery of a cellular therapy by application of relevant GMP regulations. This presentation reviews aspects of this program and chronicles our experience which at present amounts to over 4400 infusions for over 700 patients. This program provides a high degree of assurance that a cellular therapy program can be carried out in a multisite mode involving hundreds of patients through the strict adherence to cGMP as set forth in existing regulations. It would be prudent that developers of cellular andex vivo gene therapies establish a similar cell processing and QA/QC infrastructure at an early developmental stage to optimize safety and reproducibility and facilitate regulatory review.     

生态环境对川芎产、质量的影响

生态环境对川芎产、质量的影响丁德蓉,陈兴福,卢进,黄文秀,刘岁荣（四川省中医药研究院药物种植研究所,南川６４８４０８）ＩｎｆｌｕｅｎｃｅｏｆＥｃｏｅｎｖｉｒｏｎｍｅｎｔｏｎｔｈｅＹｉｅｌｄａｎｄＱｕａｌｉｔｙｏｆＬｉｇｕｓｔｉｃｕｍｃｈｕａｎｘｉｏｎ...     

植物激素与营养液配施对平菇产量和品质的影响

不同配比的ＧＡ＿３和ＫＴ与营养液配施,对平菇菌丝生长影响不同。ＧＡ＿３和ＫＴ的适当配比,能促进菌丝分枝生长,也能显著提高平菇子实体的产量和品质,但维生素Ｃ的含量有所下降。ＧＡ＿３和ＫＴ若与营养液配合施用,比单独施用更能发挥其促进作用。     

长白山站的研究数据库管理系统

长白山站的研究数据库管理系统翟永华,赵士洞（中国科学院沈阳应用生态研究所１１００１５）Ｄａｔｅ－ＢａｓｅＭａｎａｇｅｍｅｎｔＳｙｓｔｅｍｉｎＣｈａｎｇｂａｉｓｈａｎＦｏｒｅｓｔＥｃｏｓｙｓｔｅｍＲｅｓｅａｒｃｈＳｔａｔｉｏｎ．￥ＺｈａｉＹｏｎｇｈｕａ...     

Resource quality and spatial variation in sex ratios of a free-living solitary sawfly,Dineura virididorsata (Hym., Tenthredinidae)

The sex ratio in final-instar larvae of a birch-feeding, free-living solitary sawfly, Dineura virididorsata, was investigated in Finnish Lapland. The prepupal proportion of females, pooled over ten sites, was 56%, and at four individual sites the sex ratio was significantly female-biased. Larval survival from egg to prepupae did not differ between the sexes. This suggests a femalebiased primary sex ratio in the field. The sex ratio varied among the sites but not among host trees within sites. Contrary to previous results with hymenopterans, we did not find that differences in the sex ratio depended on forage quality: site-specific or tree-specific sex ratios did not correlate with the average prepupal weight. A literature search indicated that female-biased sex ratios are also common in other free-living sawflies. We are unable to explain sex ratios of Dineura virididorsata or other free-living sawflies with existing general models.     

Importance of fungi in the diet of Gammarus pulex and Asellus aquaticus

An important component of the interaction between macroinvertebrates and leaf litter in streams in the extent to which consumers can differentiate between undecomposed and decomposing leaves. The detritivores Gammarus pulex and Asellus aquaticus fed preferentially on conditioned rather on unconditioned leaf material. Growth in A. aquaticus was significantly reduced when unconditioned leaves were provided, but in G. pulex no significant effect of conditioning on growth was observed. The capacity of G. pulex to tolerate reductions in food quality seems to be a consequence of a compensatory system in which respiration rates change to compensate for reductions in food quality. In this way a constant growth rate is maintained. Increases in ingestion rates to compensate for low quality food were not observed.     

Light quality during growth of Tradescantia albiflora regulates photosystem stoichiometry, photosynthetic function and susceptibility to photoinhibition

Tradescantia albiflora (Kunth) was grown under two different light quality regimes of comparable light quantity: in red + far-red light absorbed mainly by photosystem I (PSI light) and yellow light absorbed mainly by photosystem II (PSII light). The composition, function and ultrastructure of chloroplasts, and photoinhibition of photosynthesis in the two types of leaves were compared. In contrast to regulation by light quantity (Chow et al. 1991. Physiol. Plant. 81: 175–182), light quality exerted an effect on the composition of pigment complexes, function and structure of chloroplasts in Tradescantia: PSII light-grown leaves had higher Chl a/b ratios, higher PSI concentrations, lower PSII/PSI reaction centre ratios and less extensive thylakoid stacking than PSI light-grown leaves. Light quality triggered modulations of chloroplast components, leading to a variation of photosynthetic characteristics. A larger proportion of primary quinone acceptor (Q_A) in PSI light-grown leaves was chemically reduced at any given irradiance. It was also observed that the quantum yield of PSII photochemistry was lower in PSI light-grown leaves. PSI light-grown leaves were more sensitive to photoinihibition and recovery was slower compared to PSII light-grown leaves, showing that the PSII reaction centre in PSI light-grown leaves was more easily impaired by photoinhibition. The increase in susceptibility of leaves to photoinhibition following blockage of chloroplast-encoded protein synthesis was greater in PSII light-grown leaves, showing that these leaves normally have a greater capacity for PSII repair. Inhibition of zeaxanthin formation by dithiothreitol slightly increased sensitivity to photoinhibition in both PSI and PSII light-grown leaves.     

Improving the mineral reserves and protein quality of maize (Zea mays L.) kernels using unique genes

The effects of the maize genes, o ₂ and Mal, on the concentrations of mineral nutrient cations and amino acid levels in mature maize (Zea mays L) kernels of various inbred lines were studied. Previously, the o ₂ gene has been used to improve the protein quality and increase the mineral nutrient content of kernels from some inbred lines. Genotypes possessing the Mal (multiple aleurone layer) gene, contain more than one row of aleurone cells in their kernels and this gene enhances the effect of the o ₂gene on improving kernel protein quality. Incorporating these genes into the maize genome increased accumulation of several mineral nutrients (including Ca, Mg, Zn, Fe, Mn, Zn and Cu) in some of the experimental lines studied. The physiological basis for this increase of mineral nutrients in the kernels is discussed. The effect of the Mal gene on the kernel amino acid composition and protein quality was also examined. Possibly, these genes could be used in combination in breeding programs to improve kernel quality and nutritional value of maize.