Intercalation of proflavine and a platinum derivative of proflavine into double-helical Poly(A)

The equilibria and kinetics of the interactions of proflavine (PR) and its platinum-containing derivative [PtCl(tmen)(2)HNC(13)H(7)(NHCH(2)CH(2))(2)](+) (PRPt) with double-stranded poly(A) have been investigated by spectrophotometry and Joule temperature-jump relaxation at ionic strength 0.1 M, 25 degrees C, and pH 5.2. Spectrophotometric measurements indicate that base-dye interactions are prevailing. T-jump experiments with polarized light showed that effects due to field-induced alignment could be neglected. Both of the investigated systems display two relaxation effects. The kinetic features of the reaction are discussed in terms of a two-step series mechanism in which a precursor complex DS(I) is formed in the fast step, which is then converted to a final complex in the slow step. The rate constants of the fast step are k(1) = (2.5 +/- 0.4) x 10(6) M(-1) s(-1), k(-1) = (2.4 +/- 0.1) x 10(3) s(-1) for poly(A)-PR and k(1) = (2.3 +/- 0.1) x 10(6) M(-1) s(-1), k(-1) = (1.6 +/- 0.2) x 10(3) s(-1) for poly(A)-PRPt. The rate constants for the slow step are k(2) = (4.5 +/- 0.5) x 10(2) s(-1), k(-2) = (1.7 +/- 0.1) x 10(2) s(-1) for poly(A)-PR and k(2) = 9.7 +/- 1.2 s(-1), k(-2) = 10.6 +/- 0.2 s(-1) for poly(A)-PRPt. Spectrophotometric measurements yield for the equilibrium constants and site size the values K = (4.5 +/- 0.1) x 10(3) M(-1), n = 1.3 +/- 0.5 for poly(A)-PR and K = (2.9 +/- 0.1) x 10(3) M(-1), n = 2.3 +/- 0.6 for poly(A)-PRPt. The values of k(1) are similar and lower than expected for diffusion-limited reactions. The values of k(-1) are similar as well. It is suggested that the formation of DS(I) involves only the proflavine residues in both systems. In contrast, the values of k(2) and k(-2) in poly(A)-PRPt are much lower than in poly(A)-PR. The results suggest that in the complex DS(II) of poly(A)-PRPt both proflavine and platinum residues are intercalated. In addition, a very slow process was detected and ascribed to the covalent binding of Pt(II) to the adenine.     

Clonal analysis of the development of the barley (Hordeum vulgare L.) leaf using periclinal chlorophyll chimeras

Barley seedlings that show mosaic expression of chlorophyll were selected from the progenies of mutagenized seeds. The sectored plants were grown under conditions that lead to the formation of lateral tillers, and a fraction of these had different kinds of leaf variegation. These sectorially and periclinally chimeric shoots were used to analyze the cellular organization of the barley shoot apex and the clonal development of the leaf. The shoot apex is organized in two cell lineages, L1 and L2. As well as giving rise to the epidermis, the L1 layer contributes to leaf mesophyll, particularly at the margins, but, on the adaxial side of leaf laminae, also in more central positions. The L1 layer alone is responsible for the formation of the hood, a flower homologue structure present in strains homozygoous for the dominant allele at the K (hooded ) locus. The relative contribution of L2 to leaf formation decreases in younger tillers and during tiller development from the basal to the flag leaf. Chimerism of the plants was generated by non-transmissible somatic events or by nuclear mutations. Received: 28 May 1998 / Accepted: 20 July 1998     

Isolation and characterization of potato diploid clones generating a high frequency of monohaploid or homozygous diploid androgenetic plants

Summary Cultured microspores of diploid potato clones lead with high frequency to diploid regenerants. In this paper we report on the genetic variability for in-vitro monohaploid production from anthers of diploid plants. Three diploid genotypes have been isolated which combine the capacity to regenerate monohaploid plants with outstanding embryoid production. A trait of the anther-donor clones associated with the generation of monohaploid plants is the low production of 2n pollen grains. When present in anthers of diploid genotypes, diploid unreduced microspores are, in fact, derived mainly from a first division restitution mechanism leading to high heterozygosity of the derived embryoids, a state which apparently supports superior growth in-vitro. Also, reduced microspores have been found capable of generating diploid regenerants and the adoption of the RFLP technique allowed the isolation of such diploid plants, which can be considered to be pure lines. Donor clones with a low capacity to generate monohaploids are, as expected, poor producers of homozygous diploid plants. The selection of an anther donor producing a sufficient number of monohaploid or homozygous diploid regenerants fulfills the requirements of the first part of the analytical breeding scheme, i.e., the production of homozygous diploid clones.     

Have we overestimated the benefit of human(ized) antibodies?

The infusion of animal-derived antibodies has been known for some time to trigger the generation of antibodies directed at the foreign protein as well as adverse events including cytokine release syndrome. These immunological phenomena drove the development of humanized and fully human monoclonal antibodies. The ability to generate human(ized) antibodies has been both a blessing and a curse. While incremental gains in the clinical efficacy and safety for some agents have been realized, a positive effect has not been observed for all human(ized) antibodies. Many human(ized) antibodies trigger the development of anti-drug antibody responses and infusion reactions. The current belief that antibodies need to be human(ized) to have enhanced therapeutic utility may slow the development of novel animal-derived monoclonal antibody therapeutics for use in clinical indications. In the case of murine antibodies, greater than 20% induce tolerable/negligible immunogenicity, suggesting that in these cases humanization may not offer significant gains in therapeutic utility. Furthermore, humanization of some murine antibodies may reduce their clinical effectiveness. The available data suggest that the utility of human(ized) antibodies needs to be evaluated on a case-by-case basis, taking a cost-benefit approach, taking both biochemical characteristics and the targeted therapeutic indication into account.Key words: immunogenicity, human anti-mouse antibody, cytokine release syndrome     

Multiplex-fluorescence in situ hybridization for chromosome karyotyping

Multiplex-fluorescence in situ hybridization (M-FISH) was initially developed to stain human chromosomes--the 22 autosomes and X and Y sex chromosomes--with uniquely distinctive colors to facilitate karyotyping. The characteristic spectral signatures of all different combinations of fluorochromes are determined by multichannel image-analysis methods. Advantages of M-FISH include rapid analysis of metaphase spreads, even in complex cases with multiple chromosomal rearrangements, and identification of marker chromosomes. The M-FISH technology has been extended to other species, such as the mouse. Furthermore, in addition to painting probes, the method has been used with a variety of region-specific probes. M-FISH has even recently been used for 3D studies to analyze the distribution of human chromosomes in intact and preserved interphase nuclei. Hence, M-FISH has evolved into an essential tool for both clinical diagnostics and basic research. In this protocol, we describe how to use M-FISH to karyotype chromosomes, a procedure that takes approximately 14 d if new M-FISH probes have to be generated and 3 d if the M-FISH probes are ready to use.     

An improved mRFP1 adds red to bimolecular fluorescence complementation

Protein-protein interactions are fundamental to virtually every aspect of cellular functions. Blue, green and yellow bimolecular fluorescence complementation (BiFC) systems based on GFP and its variants allow the investigation of protein-protein interactions in vivo. We have developed the first red BiFC system based on an improved monomeric red fluorescent protein (mRFP1-Q66T), expanding the range of possible applications for BiFC.     

Coimmunopurification of phosphorylated bacterial- and plant-type phosphoenolpyruvate carboxylases with the plastidial pyruvate dehydrogenase complex from developing castor oil seeds

The phosphoenolpyruvate carboxylase (PEPC) interactome of developing castor oil seed (COS; Ricinus communis) endosperm was assessed using coimmunopurification (co-IP) followed by proteomic analysis. Earlier studies suggested that immunologically unrelated 107-kD plant-type PEPCs (p107/PTPC) and 118-kD bacterial-type PEPCs (p118/BTPC) are subunits of an unusual 910-kD hetero-octameric class 2 PEPC complex of developing COS. The current results confirm that a tight physical interaction occurs between p118 and p107 because p118 quantitatively coimmunopurified with p107 following elution of COS extracts through an anti-p107-IgG immunoaffinity column. No PEPC activity or immunoreactive PEPC polypeptides were detected in the corresponding flow-through fractions. Although BTPCs lack the N-terminal phosphorylation motif characteristic of PTPCs, Pro-Q Diamond phosphoprotein staining, immunoblotting with phospho-serine (Ser)/threonine Akt substrate IgG, and phosphate-affinity PAGE established that coimmunopurified p118 was multiphosphorylated at unique Ser and/or threonine residues. Tandem mass spectrometric analysis of an endoproteinase Lys-C p118 peptide digest demonstrated that Ser-425 is subject to in vivo proline-directed phosphorylation. The co-IP of p118 with p107 did not appear to be influenced by their phosphorylation status. Because p118 phosphorylation was unchanged 48 h following elimination of photosynthate supply due to COS depodding, the signaling mechanisms responsible for photosynthate-dependent p107 phosphorylation differ from those controlling p118's in vivo phosphorylation. A 110-kD PTPC coimmunopurified with p118 and p107 when depodded COS was used. The plastidial pyruvate dehydrogenase complex (PDC(pl)) was identified as a novel PEPC interactor. Thus, a putative metabolon involving PEPC and PDC(pl) could function to channel carbon from phosphoenolpyruvate to acetyl-coenzyme A and/or to recycle CO(2) from PDC(pl) to PEPC.