SummaryReeent studies have demonstrated that the CD3-~ subunlt of the T eell antigen reeeptor (TCR) eomplex Is involved In signal transduetlon. However, the funetlon of the remalnlng Invariant subunits, CD3-y, -ö, and e, Is still poorly understood. To examine thelr role In TCR funetlon, we have eonstrueted TCR/CD3 eomplexes devold of tunetlonal ~ subunlt and showed that they are still able to trigger the produellon of Interleukln-2 in response to antigen or superantigen. These data, together with previous results, Indleate that the TCR/ CD3 eomplex Is eomposed of at least two parallel Iransduelng unlts, made 01 the YÖE and ~ ehalns, respeetlvely, Furthermore, the analysis 01 partially truneated ~ ehains has led us to Indlvlduallze a lunetlonal domaln that may have eonstltuted the buildlng block of most of the transduelng subunlts assoelated wlth antigen reeeptors and some Fe receptors. IntroduetionThe T cell antigen receptor (TCR) is a multisubunit complex composed of the products of at least six distinct genes. The TCR a and TCR ß subunits exist as disulfidelinked heterodimers, possess short cytoplasmic tails, and contain clonally variable regions that determine the antigenic specificity of the complex. The remaining subunits, termed CD3-y, -ö, -E, -~, and -1], are invariant, noncovalently associated with the TCR aß dimer, and possess large intracytoplasmic domains thought to be responsible for coupling antigen recognition to various signal transductlon pathways. The evolutionarily related y, Ö, and e subunits are expressed as noncovalently associated ye and OE pairs (Koning et al., 1990;Blumberg et al. , 1990; De la Herra et al. , 1991), and display immunoglobulin-like extracellular domains (Gold et al., 1987). In contrast, the ~ and 1] subunlts contaln an extracellular domain of only 9 residues and constitute the prototype of a new protein family that includes the y chain of the high affinity IgE receptor (FceRI)

Nanotechnology and more particularly nanotechnology-based products and materials have provided a huge potential for novel solutions to many of the current challenges society is facing. However, nanotechnology is also an area of product innovation that is sometimes developing faster than regulatory frameworks. This is due to the high complexity of some nanomaterials, the lack of a globally harmonised regulatory definition and the different scopes of regulation at a global level. Research organisations and regulatory bodies have spent many efforts in the last two decades to cope with these challenges. Although there has been a significant advancement related to analytical approaches for labelling purposes as well as to the development of suitable test guidelines for nanomaterials and their safety assessment, there is a still a need for greater global collaboration and consensus in the regulatory field. Furthermore, with growing societal concerns on plastic litter and tiny debris produced by degradation of littered plastic objects, the impact of micro- and nanoplastics on humans and the environment is an emerging issue. Despite increasing research and initial regulatory discussions on micro- and nanoplastics, there are still knowledge gaps and thus an urgent need for action. As nanoplastics can be classified as a specific type of incidental nanomaterials, current and future scientific investigations should take into account the existing profound knowledge on nanotechnology/nanomaterials when discussing issues around nanoplastics. This review was conceived at the 2019 Global Summit on Regulatory Sciences that took place in Stresa, Italy, on 24–26 September 2019 (GSRS 2019) and which was co-organised by the Global Coalition for Regulatory Science Research (GCRSR) and the European Commission's (EC) Joint Research Centre (JRC). The GCRSR consists of regulatory bodies from various countries around the globe including EU bodies. The 2019 Global Summit provided an excellent platform to exchange the latest information on activities carried out by regulatory bodies with a focus on the application of nanotechnology in the agriculture/food sector, on nanoplastics and on nanomedicines, including taking stock and promoting further collaboration. Recently, the topic of micro- and nanoplastics has become a new focus of the GCRSR. Besides discussing the challenges and needs, some future directions on how new tools and methodologies can improve the regulatory science were elaborated by summarising a significant portion of discussions during the summit. It has been revealed that there are still some uncertainties and knowledge gaps with regard to physicochemical properties, environmental behaviour and toxicological effects, especially as testing described in the dossiers is often done early in the product development process, and the material in the final product may behave differently. The harmonisation of methodologies for quantification and risk assessment of nanomaterials and micro/nanoplastics, the documentation...

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Three distinct ribosome-inactivating proteins (RIPS) were isolated from pokeweed (Phytolacca americana). We identified and sequenced for the first time a complete cDNA encoding the pokeweed antiviral protein II (PAP II), which is expressed in the late summer leaves of pokewecd. The cDNA of PAP II consists of 1,187 nucleotides and encodes a mature protein of 285 amino acids. Its predicted amino acid sequence is only 33% similar to PAP and PAP-S. The NH2 terminal extrapeptide (25 amino acid residues) was similar but not identical to that of PAP's extrapeptide. The cDNA of PAP II was expressed in E. coli. The growth of the transformants was strongly inhibited after induction of the gene. Furthermore, PAP II, which was produced in E. coli, inhibited protein synthesis in a rabbit reticulocyte translation system. Thus, recombinant PAP II would appear to be as functional as native PAP in inhibiting protein synthesis in both prokaryotes and eukaryotes.

Studies on the biochemical mechanism of promoter inhibition or inactivation by sequence-specific promoter methylations necessitated the development of a cell-free transcription system that responded to in vitro promoter methylations. Such systems were hitherto not available. In nuclear extracts from HeLa cells, the activities of two adenovirus type 2 promoters in the nonmethylated and methylated forms were compared. The late E2A promoter in vitro methylated at three 5'-CCGG-3' (HpaH) sequences at nucleotides-215, +6, and +24, or the major late promoter in vitro methylated at nucleotide-52 in the 5'-CCGG-3' sequence or at nucleotide-13 in the 5'-GCGC-3' (HhaI) sequence exhibited strikingly lower activities than did the nonmethylated constructs or exhibited no activity at all. The designated nucleotide positions were calculated relative to the cap sites of the two promoters. Upon in vitro transcription, the methylation pattern of the E2A late promoter was shown to be stable. For the inhibitory effects by sequence-specific methylations to be elicited, circular templates had to be used, the DNA titers had to be at critical levels for each extract, and high protein concentrations had to be maintained. When a template mixture of the nonmethylated major late promoter and the 5'-CCGG-3' methylated late E2A promoter of adenovirus type 2 DNA was used, the major late promoter was active and the methylated late E2A promoter was inhibited or inactivated. Activity levels of the two different promoters could be assessed simultaneously in the same assay due to differences in lengths between the products of transcription from the late E2A and major late promoters. Thus inhibition in the cell-free system could be proven to be specific for the methylated promoter. We are currently pursuing the hypothesis that cellular factors are crucial in recognizing methylated promoters and somehow participate in their inactivation.

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Pokeweed antiviral proteins (PAP) represent a family of protein toxins isolated from various organs and at different stages of development of Phytolacca americana (pokeweed). We isolated, sequenced and characterized for the first time a complete cDNA encoding a pokeweed antiviral protein expressed in seeds. The cDNA of PAP-S consists of 1249 nucleotides and encodes a mature 262 amino acid protein. Its predicted amino acid sequence is more similar to PAP (76%) than to PAP II (31%). It is known from literature that PAP-S is more active in inhibiting protein synthesis than other members of the PAP family. Therefore, the cDNA of PAP-S was expressed in Escherichia coli and the biological activity of the recombinant protein was compared with that of PAP purified from spring leaves. In a rabbit translation system, the median inhibitory concentrations (IC 50 ) of recombinant PAP-S and native PAP were determined as 0.07 and 0.29 nM, respectively. Although the PAP-S protein in seeds is glycosylated, PAP-S can be expressed in Escherichia coli in a very active form, indicating that posttranslational modification in pokeweed does not seem to alter its ability to inhibit protein synthesis.