The complete set of genes encoding major intrinsic proteins in Arabidopsis provides a framework for a new nomenclature for major intrinsic proteins in plants

Major intrinsic proteins (MIPs) facilitate the passive transport of small polar molecules across membranes. MIPs constitute a very old family of proteins and different forms have been found in all kinds of living organisms, including bacteria, fungi, animals, and plants. In the genomic sequence of Arabidopsis, we have identified 35 different MIP-encoding genes. Based on sequence similarity, these 35 proteins are divided into four different subfamilies: plasma membrane intrinsic proteins, tonoplast intrinsic proteins, NOD26-like intrinsic proteins also called NOD26-like MIPs, and the recently discovered small basic intrinsic proteins. In Arabidopsis, there are 13 plasma membrane intrinsic proteins, 10 tonoplast intrinsic proteins, nine NOD26-like intrinsic proteins, and three small basic intrinsic proteins. The gene structure in general is conserved within each subfamily, although there is a tendency to lose introns. Based on phylogenetic comparisons of maize (Zea mays) and Arabidopsis MIPs (AtMIPs), it is argued that the general intron patterns in the subfamilies were formed before the split of monocotyledons and dicotyledons. Although the gene structure is unique for each subfamily, there is a common pattern in how transmembrane helices are encoded on the exons in three of the subfamilies. The nomenclature for plant MIPs varies widely between different species but also between subfamilies in the same species. Based on the phylogeny of all AtMIPs, a new and more consistent nomenclature is proposed. The complete set of AtMIPs, together with the new nomenclature, will facilitate the isolation, classification, and labeling of plant MIPs from other species.     

Simultaneous detection of IFN-gamma and IL-4 mRNAs using RT-PCR and time-resolved fluorometry

Time-resolved fluorometry was applied in the detection of RT-PCR amplified mRNAs for the Th1 and Th2 cell-derived cytokines interferon gamma (IFN-gamma) and interleukin (IL-)4, respectively. RNA stimulated cells was reverse transcribed and the cDNAs for the cytokine mRNAs and the constantly expressed beta-actin (beta-ACT) mRNA were simultaneously amplified in one multiplex PCR reaction. The PCR conditions were optimized to minimize mutual inhibition of individual amplifications. One of the PCR primers in each primer pair was biotinylated, and the PCR products were captured onto streptavidin-coated microtitre plates. The three PCR products were detected with three different lanthanide labelled target-specific probes in solution hybridization. IFN-gamma, IL-4 and beta-ACT were detected with europium (Eu), terbium (Tb) and samarium (Sm) labelled probes, respectively, using time-resolved fluorometry. Small cell numbers used in microtitre plate cultures were sufficient to detect cytokine messages after mitogen stimulation. This sequence-based method provides a sensitive, specific, fast and nonisotopic alternative to conventional blotting and hybridisation with radioactive probes. In addition, the multiplex fluorogenic dye detection facilitates relative quantification of target mRNAs.     

Evidence for cooperative binding of (-)Isoproterenol to rat brain beta1-adrenergic receptors

In the present study, the effects of the thiol oxidising agent diamide upon the properties of rat brain beta1-adrenergic and human platelet serotonin2A receptor recognition sites have been investigated using [3H](-)CGP-12177 (in the presence of ICI-118551) and [3H]LSD as ligands. (-)Isoprenaline inhibited [3H](-)CGP-12177 binding with nH values of 0.87, 0.67, and 0.56 for added Mg2+ concentrations of 0, 2.5, and 25 mM, respectively. Pretreatment with diamide increased the nH to above unity for the inhibition of the binding by (-)isoprenaline, without a concomitant effect on the inhibition of the binding by (-)propranolol. In contrast, diamide reduced the affinity of human platelet serotonin2A-receptors for antagonists, but did not consistently induce nH values above unity for the inhibition of antagonist binding by serotonin. These results suggest that cooperative interactions reported for cardiac muscarinic receptors may also occur for beta1-adrenergic receptors in the rat brain.     

A new dominant selection marker for transformation of Pichia pastoris to soraphen A resistance

Despite the considerable progress molecular genetics of filamentous fungi has made during the past decade, there is still an urgent need for efficiently working selectable markers for fungal transformation. Using Pichia pastoris as a host, we describe the development of a new dominant selectable marker of prokaryotic origin. This system, termed sor(R), is based upon the resistance of the bacterial enzyme acetyl-CoA carboxylase (ACCase) to the macrocyclic polyketide soraphen A, a potent inhibitor of fungal ACCase produced by the myxobacterium Sorangium cellulosum. In this study, we firstly demonstrate that the integration of a single sor(R) cassette into the P. pastoris genome confers resistance to elevated concentrations of soraphen A. Furthermore, it has been shown that the versatility of this marker can be considerably increased by splitting the sor(R) cassette, especially when successive transformations are performed on the same strain. As pronounced sensitivity to soraphen A is the rule among filamentous fungi, we expect the sor(R) marker to be a widely applicable tool for fungal transformation.     

Postreplicative recruitment of cohesin to double-strand breaks is required for DNA repair

Chromosome stability depends on accurate chromosome segregation and efficient DNA double-strand break (DSB) repair. Sister chromatid cohesion, established during S phase by the protein complex cohesin, is central to both processes. In the absence of cohesion, chromosomes missegregate and G2-phase DSB repair fails. Here, we demonstrate that G2-phase repair also requires the presence of cohesin at the damage site. Cohesin components are shown to be recruited to extended chromosome regions surrounding DNA breaks induced during G2. We find that in the absence of functional cohesin-loading proteins (Scc2/Scc4), the accumulation of cohesin at DSBs is abolished and repair is defective, even though sister chromatids are connected by S phase generated cohesion. Evidence is also provided that DSB induction elicits establishment of sister chromatid cohesion in G2, implicating that damage-recruited cohesin facilitates DNA repair by tethering chromatids.     

A comparison of scoring functions for protein sequence profile alignment

MOTIVATION: In recent years, several methods have been proposed for aligning two protein sequence profiles, with reported improvements in alignment accuracy and homolog discrimination versus sequence-sequence methods (e.g. BLAST) and profile-sequence methods (e.g. PSI-BLAST). Profile-profile alignment is also the iterated step in progressive multiple sequence alignment algorithms such as CLUSTALW. However, little is known about the relative performance of different profile-profile scoring functions. In this work, we evaluate the alignment accuracy of 23 different profile-profile scoring functions by comparing alignments of 488 pairs of sequences with identity < or =30% against structural alignments. We optimize parameters for all scoring functions on the same training set and use profiles of alignments from both PSI-BLAST and SAM-T99. Structural alignments are constructed from a consensus between the FSSP database and CE structural aligner. We compare the results with sequence-sequence and sequence-profile methods, including BLAST and PSI-BLAST. RESULTS: We find that profile-profile alignment gives an average improvement over our test set of typically 2-3% over profile-sequence alignment and approximately 40% over sequence-sequence alignment. No statistically significant difference is seen in the relative performance of most of the scoring functions tested. Significantly better results are obtained with profiles constructed from SAM-T99 alignments than from PSI-BLAST alignments. AVAILABILITY: Source code, reference alignments and more detailed results are freely available at http://phylogenomics.berkeley.edu/profilealignment/     

Mesenchymal stem/progenitor cells developed in cultures from UC blood

Background Whether umbilical cord blood (UCB) serves as a source of mesenchymal stem/progenitor cells (MSPC) is controversial. MSPC are the best candidates for cellular therapy of orthopedic skeletal tissues. In order to explore the possibility of UCB as a useful source of MSPC, we identified, expanded in culture, and characterized MSPC from UCB harvests on a large scale. Methods Mononuclear cells isolated from UCB harvests (n=411) were cultured in media supplemented with 10% FBS. MSPC-like cells cultured from each UCB harvest were expanded ex vivo by successive subcultivation. UCB harvests with a more than 1000-fold expanding capacity (n=9) were examined for surface Ag phenotypes and in vitro differentiation potentials into osteogenic, chondrogenic and adipogenic lineages. Results Ninety-five out of a total of 411 UCB units (23.1%) generated MSPC-like cells during cultivation. Nine UCB units (2.2%) yielded MSPC with more than 1000-fold expansion capacity. These cells positively expressed MSPC-related Ag, but did not express myeloid, histocompatibility or endothelial Ag. These cells also possessed multiple capacities for osteogenic, chondrogenic and adipogenic differentiation. Discussion Although the incidence of UCB harvests producing MSPC in culture was low, some of them showed a more than 1000-fold expanding capacity, which is enough in cell numbers to be an allogeneic source for cellular therapy. Our results may encourage the use of UCB as an attractive target for allogeneic cellular therapeutic options in tissue engineering.     

Top-down impact of bacterivorous nematodes on the bacterial community structure: a microcosm study

The influence of bacterivorous nematodes (Diplolaimelloides meyli, Diplolaimelloides oschei, Diplolaimella dievengatensis, Panagrolaimus paetzoldi) on the development of a bacterial community growing on decaying cordgrass detritus was studied in laboratory microcosm experiments. Cordgrass leaves were incubated on a sediment surface with a natural bacterial mixture containing bacteria from sediment, cordgrass detritus and habitat water. The four nematode species were applied separately to the microcosms; controls remained without nematodes. Samples were taken seven times over a 65-day period. The bacterial community structure was analysed by means of DGGE of the 16S rRNA genes. Multi Dimensional Scaling showed grouping of the samples per treatment. Analysis of Similarities indicated that the differences between treatments were significantly larger than differences within treatments. Our results suggest that nematodes can have a significant structuring top-down influence on the 'pool' of bacteria growing on the detritus, even at low densities. Dissimilarities were similar between all treatments. Differences in bacterial community composition within the treatments with monhysterids (D. meyli, D. oschei and D. dievengatensis) can be explained by species-specific food preferences. Panagrolaimus paetzoldi on the other hand feeds unselectively, and thus affects the bacterial community differently. A top-down effect of the nematodes on the diversity of the bacterial community was only evident under high grazing pressure, i.e. in the presence of P. paetzoldi.     

Identification of the antigenic epitopes in staphylococcal enterotoxins A and E and design of a superantigen for human cancer therapy

Monoclonal antibodies have a potential for cancer therapy that may be further improved by linking them to effector molecules such as superantigens. Tumor targeting of a superantigen leads to a powerful T cell attack against the tumour tissue. Encouraging results have been observed preclinically and in patients using the superantigen staphylococcal enterotoxin A, SEA. To further improve the concept, we have reduced the reactivity to antibodies against superantigens, which is found in all individuals. Using epitope mapping, antibody binding sites in SEA and SEE were found around their MHC class II binding sites. These epitopes were removed genetically and a large number of synthetic superantigens were produced in an iterative engineering procedure. Properties such as decreased binding to anti-SEA as well as higher selectivity to induce killing of tumour cells compared to MHC class II expressing cells, were sequentially improved. The lysine residues 79, 81, 83 and 84 are all part of major antigenic epitopes, Gln204, Lys74, Asp75 and Asn78 are important for optimal killing of tumour cells while Asp45 affects binding to MHC class II. The production properties were optimised by further engineering and a novel synthetic superantigen, SEA/E-120, was designed. It is recognised by approximately 15% of human anti-SEA antibodies and have more potent tumour cell killing properties than SEA. SEA/E-120 is likely to have a low toxicity due to its reduced capacity to mediate killing of MHC class II expressing cells. It is produced as a Fab fusion protein at approximately 35 mg/l in Escherichia coli.