An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo

pEPI-1, a vector in which a chromosomal scaffold/matrix-attached region (S/MAR) is linked to the simian virus 40 origin of replication, is propagated episomally in CHO cells in the absence of the virally encoded large T-antigen and is stably maintained in the absence of selection pressure. It has been suggested that mitotic stability is provided by a specific interaction of this vector with components of the nuclear matrix. We studied the interactions of pEPI-1 by crosslinking with cis-diamminedichloroplatinum II, after which it is found to copurify with the nuclear matrix. In a south-western analysis, the vector shows exclusive binding to hnRNP-U/SAF-A, a multifunctional scaffold/matrix specific factor. Immunoprecipitation of the crosslinked DNA-protein complex demonstrates that pEPI-1 is bound to this protein in vivo. These data provide the first experimental evidence for the binding of an artificial episome to a nuclear matrix protein in vivo and the basis for understanding the mitotic stability of this novel vector class.     

Purification of TAXI-like endoxylanase inhibitors from wheat (Triticum aestivum L.) whole meal reveals a family of iso-forms

An affinity chromatography method has been developed for purification of endoxylanase inhibitors concentrated by cation exchange chromatography from wheat whole meal and is based on immobilisation of a Bacillus subtilis family 11 endoxylanase on N-hydroxysuccinimide activated Sepharose 4 Fast Flow. When followed by high-resolution cation exchange chromatography, the purification of seven TAXIs, Triticum aestivum L. endoxylanase inhibitors was achieved so extending the number of such proteins known to date (TAXI I and II). Based on their inhibition activities against a B. subtilis family 11 and an Aspergillus niger family 11 endoxylanase, six TAXI I- and only one TAXI II-like inhibitor could be distinguished. The first type of endoxylanase inhibitor is active against both endoxylanases and the second type only has significant activity against the B. subtilis endoxylanase.     

Polylactones. 59. Biodegradable networks via ring-expansion polymerization of lactones and lactides with a spirocyclic tin initiator

Spirocyclic tin initiators were prepared by condensation of commercial hydroxyethylated pentaerythritol with Bu(2)Sn(OMe)(2). These tin-containing spirocycles served as initiators for the ring-expansion polymerization of epsilon-caprolactone, beta-D,L-butyrolactone or D,L-lactide. The in situ polycondensation of these expanded spirocycles with terephthaloyl chloride or sebacoyl chloride yielded the desired biodegradable networks with elimination of the Bu(2)Sn group. The segment length (pore size) could be controlled via the monomer-initiator ratio (M/I) of the ring-expansion polymerization. Biodegradable networks were also obtained when Sn-containing spirocyclic polylactones were polycondensed with diphenyl dichlorosilane, benzene phosphonic dichloride, and phenyl phosphoric dichloride.     

Identification of a novel family of sequence repeats among prokaryotes

The rapid increase in genomic sequences provides new opportunities for comparative genomics. In this report, we describe a novel family of repeat sequences that is present in Bacteria and Archaea but not in Eukarya. The repeat loci typically consisted of repetitive stretches of nucleotides with a length of 25 to 37 bp alternated by nonrepetitive DNA spacers of approximately equal size as the repeats. The nucleotide sequences and the size of the repeats were highly conserved within a species, but between species the sequences showed no similarity. Due to their characteristic structure, we have designated this family of repeat loci as SPacers Interspersed Direct Repeats (SPIDR). The SPIDR loci were identified in more than forty different prokaryotic species. Individual species such as Mycobacterium tuberculosis contain one SPIDR locus, while other species such as Methanococcus jannaschii contained up to 20 different loci. The number of repeats in a locus varies greatly from two repeats to several dozens of repeats. The SPIDR loci were flanked by a common 300-500-bp leader sequence, which appeared to be conserved within a species but not between species. The SPIDR locus of M. tuberculosis is extensively used for strain typing. The finding of SPIDR loci in other prokaryotes, including the pathogens Salmonella, Campylobacter, and Pasteurella may extend this surveillance to other species.     

Manipulation of CD45 antigen in transplantation tolerance

CD45 is known to have tyrosine phosphatase activity for signal transduction of T cells. Immunomodulation of CD45 has been tried to prevent T cell-mediated graft rejection in organ transplantation. In vitro study showed that blockade of CD45RB, an alternative splicing isoform of CD45, inhibited proliferative response of T cells after allogeneic stimulation. Treatment with a monoclonal antibody (mAb) against CD45RB induced long-term allograft acceptance in some mouse organ transplantation models. In a rat heart allograft model, a single injection of anti-rat CD45 (RT7) mAb which bound to allomorphic region of RT7 also induced allograft acceptance. CD45/RT7 is also a useful tool of targeting hematopoietic cells, because of the selective expression on all hematopoietic cells. There are two allomorphic forms of CD45 (RT7a and RT7b) in the rat. Using RT7 system, a rat heart allograft model from RT7a donors to RT7b recipients was designed to test functional relevance of graft-associated hematopoietic cells (microchimerism) to allograft acceptance. Then donor-derived hematopoietic cells were selectively depleted using anti-RT7a mAb in vivo. Depletion on day 0 prevented allograft acceptance and was associated with severe acute or chronic graft rejection, while depletion on day 18 after transplantation showed no effect. This experimental study showed a crucial role of microchimerism in induction phase of allograft acceptance. In conclusion, the CD45/RT7 system is not only a target molecule for tolerance induction, but also an useful tool for experimental models in transplantation immunology. In this review, we introduce basic properties of CD45 and recent results with manipulation of CD45.     

High-resolution peptide mapping of cerebrospinal fluid: a novel concept for diagnosis and research in central nervous system diseases

Peptides, such as many hormones, cytokines and growth factors play a central role in biological processes. Furthermore, as degradation products and processed forms of larger proteins they are part of the protein turnover. Thus, they can reflect disease-related changes in an organism's homeostasis in several ways. Since two-dimensional gel electrophoresis is restricted to analysis and display of proteins with relative molecular masses above 5000, we developed Differential Peptide Display (DPD), a new technology for analysis and visualization of peptides. Here we describe its application to cerebrospinal fluid of three subjects without a disease of the central nervous system (CNS) undergoing routine myelography and of two patients suffering from a primary CNS lymphoma. Peptides with a relative molecular mass below 20000 were extracted and analysed by a combination of chromatography and mass spectrometry. The peptide pattern of a sample was depicted as a multi-dimensional peptide mass fingerprint with each peptide's position being characterized by its molecular mass and chromatographic behaviour. Such a fingerprint of a CNS sample consists of more than 6000 different signals. Data analysis of peptide patterns from patients with CNS lymphoma compared to controls revealed obvious differences regarding the peptide content of the samples. By analysing peptides within a mass range of 750-20000, DPD extends 2D gel electrophoresis, thus offering the chance to investigate CNS diseases on the level of peptides. This represents a new approach for diagnosis and possible therapy.     

New approaches for high-yield purification of Müllerian inhibiting substance improve its bioactivity

We have established a new method to purify Müllerian inhibiting substance (MIS) with higher purity and recovery over existing procedures. Recombinant human MIS was expressed in Chinese hamster ovary cells and secreted into chemically defined serum-free media. The secreted products were concentrated by either precipitation with ammonium sulfate or lectin-affinity chromatography, each of which was followed by anion-exchange chromatography. Further separation of the active carboxy-terminal domain of MIS was achieved after cleavage by plasmin followed by lectin-affinity chromatography. This method may be applicable to other members of the transforming growth factor beta family with which MIS shares sequence homology.     

Identification of new pisatin demethylase genes (PDA5 and PDA7) in Nectria haematococca and non-Mendelian segregation of pisatin demethylating ability and virulence on pea due to loss of chromosomal elements

Previous studies have shown that high virulence on pea in Nectria haematococca Mating Population VI is linked to the ability to detoxify the pea phytoalexin, pisatin, via demethylation (Pda). To test this linkage further, a highly virulent Pda(+) isolate (34-18) was used as the recurrent parent in backcrosses to Pda(-) isolates, but most of the progeny were low in virulence on pea, and tetrad analysis gave conflicting ratios for the genetic control of Pda. Southern analysis of 34-18 and progeny showed that 34-18 carries a gene similar to PDA1 (PDA1-2), two new PDA genes, PDA5 and PDA7, and that all three genes can be lost during meiosis. Southern analysis of electrophoretic karyotypes showed that PDA1-2 is on a 1.5-Mb dispensable chromosome in 34-18 and that PDA5 and PDA7 are on a 4.9-Mb chromosome in 34-18 but are found on variably sized chromosomes in progeny. Loss of PDA5 or PDA7 in progeny was not generally associated with morphological phenotypes, except in progeny from some crosses between PDA5 parents. Loss of PDA5 was associated with growth abnormalities in these crosses, suggesting that in some genetic backgrounds at least a portion of the PDA5/PDA7 chromosome is essential for normal growth. All highly virulent progeny had PDA1-2 or a combination of PDA5 and PDA7 while isolates that lacked the three genes were low in virulence, supporting the hypothesis that Pda, or genes linked to PDA genes, are necessary for virulence on pea. However, low virulence isolates with PDA genes were also identified, suggesting that there are pathogenicity genes that can segregate independently of PDA genes.