Donor height in combination with islet donor score improves pancreas donor selection for pancreatic islet isolation and transplantation.

To maximize the islet isolation yield for successful islet transplantation, the key task has been to identify an ideal pancreas donor. Since implementation of the islet donor score in donor selection, we have consistently obtained higher islet yields and transplantation rates. In this study, we tested whether assessing donor height as an independent variable in combination with the donor score could improve the pancreas donor selection. Donor and islet isolation information (n = 22) were collected and studied between 2011 and 2012. Pearson correlation analysis was used in statistical analysis. Donor height as an independent variable was significantly correlated to the weight of the pancreas, pre-Islet Equivalents (pre-IEQ), post-IEQ, and IDS (P < .05). When donor with height of 179 cm ± 3 was selected in combination with IDS > 80, the clinical islet transplantation rate reached 80%.

Ras and p53 intracellular targeting with recombinant single-chain Fv (scFv) fragments: a novel approach for cancer therapy?

Intracellular expression of recombinant antibodies allows one to interfere with the functions of oncogenic molecules expressed in various cell compartments and has therefore a vast clinical potential in cancer therapy. We inhibited the functions of oncogenic Ras mutant forms by intracellular expression of a neutralizing single-chain antibody (scFv). In vitro studies indicated that the scFv is expressed in the cytosol of Xenopus laevis oocytes and of tumor cells, blocks ras-mediated activation processes, and induces tumor cell death. In vivo studies performed using scFv cDNA inserted into an adenoviral vector showed that the scFv dramatically affects tumor growth. Second, intracellular expression of scFvs directed against p53 indicated that these antibody fragments can be successfully targeted to cell nucleus, bind p53, and partially restore the transcriptional activity of p53 mutants in human tumor cells. Thus, intracellular scFvs directed against oncogenic molecules may represent a new class of antitumor agents.

Carcinoembryonic antigen (CEA)-specific T-cell activation in colon carcinoma induced by anti-CD3 x anti-CEA bispecific diabodies and B7 x anti-CEA bispecific fusion proteins.

Two bispecific recombinant molecules, an anti-CD3 x anti-carcinoembryogenic antigen (CEA) diabody and a B7 x anti-CEA fusion protein, were tested for their capacity to specifically activate T cells in the presence of CEA-expressing colon carcinoma cells. T-cell activation by the anti-CD3 x anti-CEA diabody required close contact to CEA-positive cells and resulted in diabody-mediated cytotoxicity against the target cells. Additionally, CD28-mediated costimulation in combination with anti-CD3 x anti-CEA diabodies induced activation of autologous T cells in CEA-positive primary colon carcinoma specimens, as determined by flow cytometry. The high specificity of the bispecific diabody approach could be further enhanced by the use of B7 x anti-CEA fusion proteins because the costimulatory CD28-signaling to the T cells strictly depended on the expression of CEA on the target cells. We demonstrate that displaying engagement sites for the T-cell antigens CD3 and CD28 on the surface of colon carcinoma cells is a suitable way to activate and retarget T cells in a highly tumor-specific manner. For clinical purposes, B7 x anti-tumor-associated antigen (TAA) fusion proteins, which are equally effective but more specific compared with anti-CD28 monoclonal anti-bodies, thus may improve the tumor specificity of anti-CD3 x anti-TAA bispecific antibodies. Furthermore, B7-negative tumors can be converted into B7-positive tumors by B7 x anti-TAA fusion proteins without the need for B7 gene transfer to the malignant cells.

Interaction of elongation factor eEF-2 with ribosomal P proteins.

The eukaryotic P1 and P2 ribosomal proteins which constitute, with P0, a pentamer forming the lateral stalk of the 60 S ribosomal subunit, exhibit several differences from their prokaryotic equivalents L7 and L12; in particular, P1 does not have the same primary structure as P2 and both of them are phosphorylated, the significance of the latter remaining unclear. Rat liver P1 and P2 were overproduced in Escherichia coli cells and their interaction with elongation factor eEF-2 was studied. Both recombinant proteins were found to be required for the ribosome-dependent GTPase activity of eEF-2, with P2 in the phosphorylated form. The surface plasmon resonance technique revealed that, in vitro, both proteins interact specifically with eEF-2, with a higher affinity for P1 (Kd = 3.8 x 10-8 m) than for P2 (Kd = 2.2 x 10-6 m). Phosphorylation resulted in a moderate increase (two- to four-fold) in these affinities. The interaction of both P1 and P2 (phosphorylated or not) with eEF-2 resulted in a conformational change in the factor, revealed by an increase in the accessibility of Glu554 to proteinase Glu-C. This increase was observed in both the presence and absence of GTP and GDP, which themselves produced marked opposite effects on the conformation of eEF-2. Our results suggest that the two proteins P1 and P2 both interact with eEF-2 inducing a conformational transition of the factor, but have acquired some specific properties during evolution.

Mutations in the C-terminal domain of topoisomerase II affect meiotic function and interaction with the casein kinase 2 beta subunit.

Topoisomerase II is a major target of the protein kinase casein kinase 2 (PK CK2) in vivo. All major phosphorylation acceptor sites in the yeast enzyme are found in the C-terminal 350aa. The acceptor sites are generally clustered such that there is more than one modified Ser or Thr within a short peptide. Mutagenesis of the predicted acceptor sites have confirmed that five of the eight predicted sites are targeted in vitro and in vivo by PK CK2. Mutation to nonphosphorylatable, neutral residues provokes at most a 10% increase in mitotic doubling time. Truncation of the enzyme leaves the enzyme catalytically active, but slightly lengthens the doubling time during mitotic growth and impedes progress through meiosis. Since this could reflect the loss of interaction with an important ligand, we have examined whether the C-terminal domain of the yeast enzyme mediates interaction with the regulatory beta subunit of PK CK2, which was previously reported to bind topoisomerase II. We find that point mutation of the phospho-acceptor sites does not abrogate the interaction with a small region of PK CK2beta, while truncation at aa1276 or aa1236 does. The site of interaction within PK CK2beta does not coincide with the highly negatively charged spermine binding site.

The binding of the alpha subunit of protein kinase CK2 and RAP74 subunit of TFIIF to protein-coding genes in living cells is DRB sensitive.

In a previous report, we documented that a major portion of the nuclear protein kinase CK2alpha (CK2alpha) subunit does not form heterooligomeric structures with the beta subunit, but it binds tightly to nuclear structures in an epithelial Chironomus cell line. We report here that the CK2alpha, but not beta, subunit is co-localized with productively transcribing RNA polymerase II (pol II) on polytene chromosomes of Chironomus salivary gland cells. Likewise, the RAP74 subunit ofTFIIF, a potential substrate for CK2, is co-localized with pol II. The occupancies of chromosomes with the CK2alpha and RAP74 subunits are sensitive to DRB, an inhibitor of pol II-based transcription and the activity of CK2 and pol II carboxyl-terminal kinases. DRB alters the chromosomal distribution of the CK2alpha and RAP74 subunits: there is a time-dependent clearance from the chromosomes of CK2alpha and RAP74 subunits, which coincides in time the completion and release of preinitiated transcripts after addition of DRB. The results suggest that both the CK2alpha and RAP74 subunits travel with the elongating pol II molecules along the DNA template during the entire transcription cycle. No detectable re-association of CK2alpha and RAP74 with the promoters takes, however, place after the completion of the preinitiated transcripts in the presence of DRB. In contrast, the binding of hypophosporylated pol II and TFIIH to the active gene loci is not abolished by the DRB regimen. Our data are consistent with the possibility that in living Chironomus salivary gland cells, DRB interferes with the recruitment of TFIIF, but not of TFIIH, to the promoter by interference with the activity of the CK2alpha subunit enzyme and phosphorylation of RAP74 and thereby DRB blocks transcription initiation.

A tumor specific single chain antibody dependent gene expression system.

The design of conditional gene expression systems restricted to given tissues or cellular types is an important issue of gene therapy. Systems based on the targeting of molecules characteristic of the pathological state of tissues would be of interest. We have developed a synthetic transcription factor by fusing a single chain antibody (scFv) directed against p53 with the bacterial tetracycline repressor as a DNA binding domain. This hybrid protein binds to p53 and can interact with a synthetic promoter containing tetracycline-operator sequences. Gene expression can now be specifically achieved in tumor cells harboring an endogenous mutant p53 but not in a wild-type p53 containing tumor cell line or in a non-transformed cell line. Thus, a functional transactivator centered on single chain antibodies can be expressed intracellularly and induce gene expression in a scFv-mediated specific manner. This novel class of transcriptional transactivators could be referred as 'trabodies' for transcription-activating-antibodies. The trabodies technology could be useful to any cell type in which a disease related protein could be the target of specific antibodies.

Intracellular expression of an antibody fragment-neutralizing p21 ras promotes tumor regression.

Mutated ras genes are found in a large number of human tumors and, therefore, constitute one of the primary targets for cancer treatment. Microinjection of the neutralizing anti-Ras monoclonal antibody Y13-259 was previously reported to induce transient phenotypic reversion of ras-transformed rodent fibroblasts in vitro. We have prepared a single-chain Fv fragment (scFv) derived from Y13-259, and here, we show that intracellular expression of the scFv led to the specific inhibition of the Ras signaling pathway in Xenopus laevis oocytes and NIH3T3 fibroblasts. Moreover, neutralizing Ras with the scFv specifically promoted apoptosis in vitro in human cancer cells but not in untransformed cells. As a step toward cancer gene therapy, we finally demonstrated that intratumor transduction of HCT116 colon carcinoma cells with the anti-Ras scFv using an adenoviral vector elicited sustained tumor regression in nude mice.

Intracellular expression and functional properties of an anti-p21Ras scFv derived from a rat hybridoma containing specific lambda and irrelevant kappa light chains.

A rat single-chain Fv (Y238 scFv) was derived from the Y13-238 monoclonal antibody, a non-neutralizing anti-Ras antibody. The Y13-238 hybridoma expresses two functional light chains. N-terminus microsequencing of these chains showed the presence of the Y3 Ag1.2.3 Vkappa chain derived from the rat fusion partner and of a rat Vlambda chain. Primers designed for rat Vlambda amplification allowed the cloning of a functional scFv that could bind p21Ras. The kinetics of interaction of purified Y238 scFv with the p21Ras protein was evaluated by BIAcore with a NTA sensor chip and gave an apparent affinity constant in the nanomolar range (K(D)=4.58+/-0.63 nM). Immunoprecipitation experiments of Y238 scFv expressed in Xenopus laevis oocytes confirmed the specificity of the scFv for the Ras protein. Y238 scFv could be intracellularly expressed in oocytes and in mammaliam cells without adverse effect on the Ras signalling cascade. This scFv was therefore used as control in experiments where another anti-Ras scFv (Y259 scFv, derived from the neutralizing anti-Ras mAb Y13-259) blocked the Ras pathway in vitro and led to tumor regression in a nude mouse model [Cochet, O., Kenigsberg, M., Delumeau, I., Virone-Oddos, A., Multon, M.C., Fridman, W.H., Schweighoffer, F., Teillaud, J.L., Tocqué, B., 1998. Intracellular expression of an antibody fragment-neutralizing p21 ras promotes tumor regression. Cancer Res. 58, 1170-1176.]. Finally, BIAcore analyses indicated that the epitopes recognized by Y238 and Y259 scFvs are not overlapping and allowed a more precise definition of the Y13-238 epitope.

Making hybrids of two-hybrid systems.

Two-hybrid systems are powerful tools to find new partners for a protein of interest. However, exchange of material between two-hybrid users has been handicapped by the various versions of two-hybrid systems available and by the widely accepted idea that they are not compatible. In the present paper we show that, contrary to the dogma, the most often used two-hybrid systems may be combined by either transformation or mating assays. The protocol to be followed in each case is provided. This will greatly increase the prospects of the growing network of interacting proteins, by reconciling the "two-hybrid systems" and the "interaction trap".

A new set of monoclonal antibodies against human Fc gamma RII (CD32) and Fc gamma RIII (CD16): characterization and use in various assays.

Four mouse anti-human Fc gamma RII (CD32) (6C4, 2B2, 3D3, 93.4) (IgG1, kappa) and one anti-human Fc gamma RIII (CD16) (7.5.4) IgG1, kappa) MAbs were raised. An in vitro switch variant, 7.5.4Sw50 (IgG2b, kappa), was also derived from the 7.5.4 MAb. 6C4, 2B2, and 3D3 MAbs bind both Fc gamma RIIa and Fc gamma RIIb isoforms. Two of them (6C4 and 2B2 MAbs) allow a complete blockade of the binding of immune complexes to Fc gamma RII. All three MAbs immunoprecipitate the receptor and bind both its glycosylated and nonglycosylated forms. The fourth anti Fc gamma RII MAb, 93.4, directed against the intracellular region of Fc gamma RIIa1/2, allows its detection by Western blotting only when it is not phosphorylated. The 7.5.4 MAb binds both Fc gamma RIIIa and Fc gamma RIIIb, can be used in Western blotting and does not inhibit aggregated IgG binding. ELISA using IV.3 (anti-Fc gamma RIIa1/2)/6C4 and 3G8 (anti-Fc gamma RIIIa/b)/7.5.4Sw50 MAb pairs make it possible to detect soluble Fc gamma RIIa1/2 and Fc gamma RIII, with a sensitivity of 200 pg/mL and 1 ng/mL, respectively. Surface plasmon resonance analyses indicated that the KD of two of the three anti-Fc gamma RII and of the anti-Fc gamma RIII are in the same order of magnitude (6C4: 0.78 nM, 2B2: 0.28 nM, 7.5.4: 0.47 nM). The anti-Fc gamma RII 3D3 MAb exhibits an off-rate constant higher than the 6C4 and 2B2 MAbs and a KD of 2.19 nM.

Epitope mapping and tight-binding inhibition with monoclonal antibodies directed against Escherichia coli glucosamine 6-phosphate synthase.

In the present work, we attempt to identify inhibitory monoclonal antibodies directed against Escherichia coli glucosamine-6P synthase (GlmS) and to localize the corresponding epitopes to better understand the topology of the enzyme during catalysis. Four of the 15 monoclonal antibodies have been shown to be specific for the native form of the enzyme and 2 of them, 505.1 and 522.2, strongly inhibit the glucosamine synthase activity. Kinetic analysis of 505.1 antibody behavior revealed a tight-binding inhibition with a Ki = 40 +/- 20 pM, a value which is four orders of magnitude lower than the best active site-directed inhibitor reported so far. The reactivity of all the monoclonal antibodies with 601 overlapping octapeptides covering the entire sequence of GlmS was tested by enzyme-linked immunosorbent assay for precise epitope mapping. Four linear epitopes specific for the denatured protein and one present in both native and denatured enzyme were defined by this approach. Neither 505.1 nor 522.2 was directed against linear epitopes. However, evidence for the binding of 505.1 at the glutamine catalytic site was shown by using site-directed mutants of GlmS as well as by competition experiments with an irreversible inhibitor. The mAb 105.1, which recognizes the octapeptide containing the sequence RWATHG conserved among the six glucosamine-6P synthases reported so far, allowed the detection of the human enzyme.

Phage libraries for generation of clinically useful antibodies.

Insertion of antibody genes into filamentous bacteriophage makes it possible to generate and screen libraries of 10(7) or more antibodies. Each phage expresses an antibody on its surface and contains the corresponding antibody gene. Genes that encode antibodies with desired characteristics are readily selected and their antibodies expressed as soluble proteins in Escherichia coli. We used this system to produce an antibody to carcinoembryonic antigen with higher affinity and better tumour specificity than antibodies currently in use.

Casein kinase 2 and the cell response to growth factors.

Different approaches have been followed with the aim of delineating a possible role of casein kinase 2 (CK2) in the mitogenic signalling in response to cell growth factors. (a) Immunocytochemical detection of CK2 showed that while the kinase is evenly distributed throughout cycle arrested cells, it becomes preferentially associated with the nuclear compartment in activity growing cells; (b) CK2 biosynthesis is activated as an early response of quiescent cells to growth factors. The newly synthesized CK2 steadily accumulates as the cells progress through the G1 phase. This growth factor-induced CK2 biosynthesis involves in parallel the two alpha and beta subunits of the kinase, with no detectable preferential subcellular localization of the newly synthesized enzyme; and (c) In addition to substrate phosphorylation, CK2 may form molecular complexes with cell components of functional significance. Such is the case with the protein p53, a major negative regulator of the cell cycle. CK2 forms a high affinity association (Kd 70 nM) with p53, through its beta subunit. The complex dissociates in the presence of adenosine triphosphate (ATP). These observations suggest that CK2 and p53 may play a coordinated regulatory role in the cell response to growth factors.

Coordinated regulation of amino sugar-synthesizing and -degrading enzymes in Escherichia coli K-12.

The intracellular concentration of the enzyme glucosamine-6-phosphate synthase, encoded by the gene glmS in Escherichia coli, is repressed about threefold by growth on the amino sugars glucosamine and N-acetylglucosamine. This regulation occurs at the level of glmS transcription. It is not due just to the presence of intracellular amino sugar phosphates, because mutations which derepress the genes of the nag regulon (coding for proteins involved in the uptake and metabolism of N-acetylglucosamine) also repress the expression of glmS in the absence of exogenous amino sugars.

Monoclonal antibodies against bacterial glucosamine 6-phosphate synthase: production and use for structural studies.

Fifteen mouse x rat hybridoma cell lines producing rat monoclonal antibodies (MAbs) directed to Escherichia coli Glucosamine 6-P Synthase (GlmS) were established and characterized. Most of them (13/15) are IgG2a while 2 were typed as IgG1. Their Kaff ranged from 1.5 x 10(6) to 9.6 x 10(8) M-1 as determined by Beatty et al. (1). The epitopes recognized by these MAbs were assigned to one of the two catalytical domains of the enzyme (CT1 and CT2) as demonstrated both by ELISA and Western-blotting using purified GlmS proteolytic fragments. The binding of the MAbs on either the native or denatured forms of GlmS, CT1 and CT2 was further analyzed by competitive immunoassay and most of the MAbs were found to bind preferentially to the denatured proteins. The study of the antigenic topography of GlmS by competitive radioimmunoassay demonstrated the existence of at least 10 independent epitopes on GlmS, divided into three groups. The first one (3/15) includes MAbs whose binding was not inhibited by any of the other MAbs. The second group (9/15) is comprised of MAbs that exhibit reciprocal binding inhibitory activity while the third group includes MAbs (3/15) presenting asymmetric inhibitory activity. Finally, since most of the isolated antibodies (10/15) bind to the 27 kDa amino-terminal glutamine binding domain (CT2), the capacity of these MAb to interfere with the associated glutaminase activity was analyzed.