Preliminary validation of an activation assay for ex vivo activated T cells utilized in cancer immunotherapy

An in vitro assay that measures the activation level of ex vivo activated (EVA) T cells currently being used in the adoptive immunotherapy of metastatic renal cell carcinoma has been developed. This assay is based on the ability of activated, but not resting. T cells to proliferate in response to the protein kinase C activator, phorbol myristate (PMA). To utilize this assay for in-process monitoring and control, we have begun an initial validation of the overall reproducibility of this assay. The proliferation of activated T cells in response to PMA, as measured by the mean cpm values of (3)H-thymidine incorporated, was demonstrated to have intra-assay coefficients of variation (cv's) for individual analysts that were typically less than 10% and rarely exceeded 20%. Activated T cells could be frozen and stored for at least 6 weeks with little or no deterioration in their ability to proliferate in response to PMA. Using these cells, inter-assay cv's that were typically less than 15% were obtained by individual analysts, and overall cv's of 10% to 25% were obtained for different samples assayed by different analysts at different times. This level of variability is very reasonable for a cellular assay. Furhter validation of this assay will address the issues of sensitivity, linearity and selectivity. To date, this assay has been used to analyze over 90 patient EVA cell samples and has revealed a broad range of proliferative responses to PMA. Taken together, these results suggest that this assay may be useful in defining the potency of the activated T cell used therapeutically.     

Some improvements in the quality of NAA procedures and the reliability of the results

After considering the need for quality control in NAA, the concept of quality in NAA procedures themselves is discussed, and some important factors identified. Two approaches to improve quality are then described in more detail. The first concerns the unique ability of NAA using different isotopic reactions and different modes (INAA/RNAA) to provide independent data sets in the same laboratory, thus allowing internal validation or crosschecking. The second discusses the need for chemical yield measurements in RNAA and the advantages of the radioisotopic tracer technique. Some recent advances and further possibilities for this use of tracers are listed.     

Developments in tomographic methods for biological trace element research

Neutron-induced γ-ray emission tomography for quantitative determination of the concentration and distribution of elements in a selected plane through a biological specimen is briefly explained and applied by way of illustration to the analysis of gallstones. A system capable of carrying out studies of the binding site of⁷⁵Se in different matrices using time differential perturbed angular correlation spectroscopy is also briefly described. Developments in the detector technology of positron emission tomography have allowed small-diameter imaging devices to be built for in vivo preclinical evaluation of new tracers in small animals and are discussed in the context of a proposed experiment combining the techniques mentioned above.     

Chloroplast targeting of spectinomycin adenyltransferase provides a cell-autonomous marker for monitoring transposon excision in tomato and tobacco

Antibiotic resistance genes can act as either cell autonomous or non-cell autonomous genetic markers with which to monitor the excision of plant transposons. To convert spectinomycin resistance from a noncell autonomous resistance to cell autonomous resistance, a transit peptide for chloroplast localization from a petunia ribulose bisphosphate carboxylase (rbcS) gene was fused in-frame to the aadA gene, which confers spectinomycin and streptomycin resistance. Constructs were generated in which the expression of this chimeric gene was prevented by the presence, in the 5 untranslated leader, of the maize transposons Activator (Ac) or Dissociation (Ds). When progeny of tobacco or tomato plants transformed with these constructs were germinated on spectinomycin-containing medium, germinally revertant and somatically variegated individuals could be distinguished.     

Two conformational states of Candida rugosa lipase.

The structure of Candida rugosa lipase in a new crystal form has been determined and refined at 2.1 A resolution. The lipase molecule was found in an inactive conformation, with the active site shielded from the solvent by a part of the polypeptide chain-the flap. Comparison of this structure with the previously determined "open" form of this lipase, in which the active site is accessible to the solvent and presumably the substrate, shows that the transition between these 2 states requires only movement of the flap. The backbone NH groups forming the putative oxyanion hole do not change position during this rearrangement, indicating that this feature is preformed in the inactive state. The 2 lipase conformations probably correspond to states at opposite ends of the pathway of interfacial activation. Quantitative analysis indicates a large increase of the hydrophobic surface in the vicinity of the active site. The flap undergoes a flexible rearrangement during which some of its secondary structure refolds. The interactions of the flap with the rest of the protein change from mostly hydrophobic in the inactive form to largely hydrophilic in the "open" conformation. Although the flap movement cannot be described as a rigid body motion, it has very definite hinge points at Glu 66 and at Pro 92. The rearrangement is accompanied by a cis-trans isomerization of this proline, which likely increases the energy required for the transition between the 2 states, and may play a role in the stabilization of the active conformation at the water/lipid interface. Carbohydrate attached at Asn 351 also provides stabilization for the open conformation of the flap.     

New technique in optical resolutions

The present paper illustrates the development of an advanced technique in optical resolution. Both of the amphetamine enantiomers can be obtained by a two-step distillation in nearly quantitative yield without any loss of the resolving agent. It is proved that the second-order interactions (H-bond) are sufficient for separation of enantiomers by distillation. © 1994 Wiley-Liss, Inc.     

γ射线诱发大鼠胚胎细胞转化与N－ras的激活

以γ射线诱发转化的大鼠胚胎细胞(REC:myc:γ33)的DNA构建粘粒基因库,用总基因库DNA转染NIH/3T3细胞,产生转化灶的DNA作二轮转染,二轮转化的NIH/3T3细胞内有大鼠REC:myc:γ33DNA中具转化活性的N-ras基因,用不对称PCR和DNA序列分析法证明,REC:myc:γ33细胞中鼠N-ras的活化是由于第61位密码子的A→G点突变.NIH/3T3转化灶中鼠N-ras也有同样点突变,但NIH/3T3细胞的内源性N-ras基因则无此突变.     

Analysis of the frequency of inheritance of transposed Ds elements in Arabidopsis after activation by a CaMV 35S promoter fusion to the Ac transposase gene

The Ac/Ds transposon system of maize shows low activity in Arabidopsis. However, fusion of the CaMV 35S promoter to the transposase gene (35S::TPase) increases the abundance of the single Ac mRNA encoded by Ac and increases the frequency of Ds excision. In the experiments reported here it is examined whether this high excision frequency is associated with efficient re-insertion of the transposon. This was measured by using a Ds that carried a hygromycin resistance gene (HPT) and was inserted within a streptomycin resistance gene (SPT). Excision of Ds therefore gives rise to streptomycin resistance, while hygromycin resistance is associated with the presence of a transposed Ds or with retention of the element at its original location. Self-fertilisation of most individuals heterozygous for Ds and 35S::TPase produced many streptomycin-resistant (strep^r) progeny, but in many of these families a small proportion of strep^r seedlings were also resistant to hygromycin (hyg^r). Nevertheless, 70% of families tested did give rise to at least one strep^r, hyg^r seedling, and over 90% of these individuals carried a transposed Ds. In contrast, the Ac promoter fusion to the transposase gene (Ac::TPase) produced fewer strep^rhyg^r progeny, and only 53% of these carried a transposed Ds. However, a higher proportion of the strep^r seedlings were also hyg^r than after activation by 35S::TPase. We also examined the genotype of strep^r, hyg^r seedlings and demonstrated that after activation by 35S::TPase many of these were homozygous for the transposed Ds, while this did not occur after activation by Ac::TPase. From these and other data we conclude that excisions driven by 35S::TPase usually occur prior to floral development, and that although a low proportion of strep^r progeny plants inherit a transposed Ds, those that do can be efficiently selected with an antibiotic resistance gene contained within the element. Our data have important implications for transposon tagging strategies in transgenic plants and these are discussed.