A new concept for DNA-arrays

We will insert a cleavage site in an oligodeoxynucleotide, which can be used for a selective and quantitative cleavage. For that reason we synthesized the four 5'-S-(4,4'-dimethoxytrityl)-mercapto-2'-deoxynucleotide-3'-O-(2-cyanoethoxydiisopropylamino)-phosphites (5a-d). The cleavage of P-S and C-S bonds is described (Mag, M.; Lücking, S.; Engels, J.W. Synthesis and selective cleavage of an oligodeoxy-nucleotide containing a bridged internucleotide 5'-phosphorthioate linkage. Nucleic Acids Res. 1991, 19 (7), 1437-1441; Marriott, J.H.; Mottahedeh, M.; Reese, C.B. 9-(4-methoxyphenyl)xanthen-9-thiol: A useful reagent for the preparation of thiols. Tetrahedron Lett. 1990, 31 (51), 7485-7488; Divakar, K.J.; Mottoh, A.; Reese, C.B.; Shanghvi, Y.S. Approaches to the synthesis of 2' thio analogues of pyrimidine ribosides. J. Chem. Sc., Perkin Trans. 1 1990, 969-974). The oligodeoxynucleotides with an achiral bridged 5'-phosphorothioate linkage 5'-O-P-S-3' are synthesized by the phosphoramidite procedure.     

P2Y(1) receptor activation inhibits NMDA receptor-channels in layer V pyramidal neurons of the rat prefrontal and parietal cortex

In the 1st part of this study, monosynaptic excitatory postsynaptic potentials (EPSPs) in layer V of the rat prefrontal cortex (PFC) were evoked by electrical stimulation of layer I. Recordings with intracellular sharp, microelectrodes showed a concentration-dependent inhibition of the EPSP by adenosine 5'-O-(2-thiodiphosphate) (ADP-beta-S). Pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), when given alone depressed the EPSP and in addition antagonized the effect of ADP-beta-S. Exclusion of the N-methyl-D-aspartate (NMDA) component of the EPSP by D(.)-amino-5-phosphonopentanoic acid (AP-5) abolished the ADP-beta-S-induced depression. The pressure-application of both NMDA and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) caused reproducible depolarizations. ADP-beta-S inhibited the effect of NMDA, but did not alter that of AMPA. PPADS was also under these conditions antagonistic with ADP-beta-S. In the 2nd part of the study, NMDA-induced currents were measured by whole-cell patch-clamp pipettes. ADP-beta-S caused a concentration-dependent inhibition of the responses to NMDA. PPADS alone did not alter the NMDA-currents but again antagonized the action of ADP-beta-S; 2'-deoxy-N(6)-methyladenosine-3',5'-diphosphate (MRS 2179) also abolished the NMDA effect. The ADP-beta-S-induced inhibition persisted in the presence of tetrodotoxin (TTX) or guanosine 5'-O-(3-thiodiphosphate) (GDP-beta-S) applied to the external medium and the pipette solution, respectively. The 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) moderately decreased the ADP-beta-S effect. The inhibitory function of ADP-beta-S on EPSPs and the interaction with PPADS was observed also in layer V pyramidal neurons of the parietal somatosensory cortex. In conclusion, metabotropic P2Y(1) receptors appear to exert a new modulatory influence on fast excitatory amino acid transmission in the cerebral cortex.     

Crystal structure of the carboxyltransferase subunit of the bacterial sodium ion pump glutaconyl-coenzyme A decarboxylase

Glutaconyl-CoA decarboxylase is a biotin-dependent ion pump whereby the free energy of the glutaconyl-CoA decarboxylation to crotonyl-CoA drives the electrogenic transport of sodium ions from the cytoplasm into the periplasm. Here we present the crystal structure of the decarboxylase subunit (Gcdalpha) from Acidaminococcus fermentans and its complex with glutaconyl-CoA. The active sites of the dimeric Gcdalpha lie at the two interfaces between the mono mers, whereas the N-terminal domain provides the glutaconyl-CoA-binding site and the C-terminal domain binds the biotinyllysine moiety. The Gcdalpha catalyses the transfer of carbon dioxide from glutaconyl-CoA to a biotin carrier (Gcdgamma) that subsequently is decarboxylated by the carboxybiotin decarboxylation site within the actual Na(+) pump (Gcdbeta). The analysis of the active site lead to a novel mechanism for the biotin-dependent carboxy transfer whereby biotin acts as general acid. Furthermore, we propose a holoenzyme assembly in which the water-filled central channel of the Gcdalpha dimer lies co-axial with the ion channel (Gcdbeta). The central channel is blocked by arginines against passage of sodium ions which might enter the central channel through two side channels.     

Iron distribution in three central Amazon tree species from whitewater-inundation areas (várzea) subjected to different iron regimes

Trees inhabiting central Amazon floodplain forests are subjected to an annual flood-pulse lasting up to 10 months, leading to both oxygen shortage and accumulation of high levels of reduced iron. To understand the mechanisms underlying the adaptation to these conditions, cuttings from three tree species typical of várzea inundation forests (Salix martiana, Tabernaemontana juruana, and Laetia corymbulosa), were cultivated either aerobically or anaerobically under different iron regimes in greenhouse experiments. Although all species are considered to be non-deciduous, Laetia corymbulosa lost and formed new leaves continuously during the experimental period. Although relative growth rates (RGRs) of all species declined in response to hypoxic conditions, no marked changes in RGRs were apparent among different iron concentrations in the growth medium, ranging from 50 to 500 µM, supplied in ferrous form as FeSO₄. Whereas roots exhibited color changes due to the formation of iron precipitates, no visual symptoms of iron toxicity were observed in the leaves. Iron concentration increased in all organs of all species with increasing iron concentrations in the medium, except for leaves of S. martiana and T. juruana, suggesting an effective restriction of iron influx into the leaf symplast. Although the leaf iron concentration was at the upper limit of the critical range at high external iron levels, it is suggested that internal active transport rather than intracellular detoxification mechanisms contribute to the tolerance to supra-optimal iron levels. Anatomical traits such as suberization of peripheral cell walls and the formation of aerenchyma appear to be of minor importance for Fe tolerance.     

Single-cell electroporation

Electroporation is a widely used method for the introduction of polar and charged agents such as dyes, drugs, DNA, RNA, proteins, peptides, and amino acids into cells. Traditionally, electroporation is performed with large electrodes in a batch mode for treatment of a large number of cells in suspension. Recently, microelectrodes that can produce extremely localized electric fields, such as solid carbon fiber microelectrodes, electrolyte-filled capillaries and micropipettes as well as chip-based microfabricated electrode arrays, have proven useful to electroporate single cells and subcellular structures. Single-cell electroporation opens up a new window of opportunities in manipulating the genetic, metabolic, and synthetic contents of single targeted cells in tissue slices, cell cultures, in microfluidic channels or at specific loci on a chip-based device.     

Glucose kinase of Streptomyces coelicolor A3(2): large-scale purification and biochemical analysis

Glucose kinase of Streptomyces coelicolor A3(2) is essential for glucose utilisation and is required for carbon catabolite repression (CCR) exerted through glucose and other carbon sources. The protein belongs to the ROK-family, which comprises bacterial sugar kinases and regulators. To better understand glucose kinase function, we have monitored the cellular activity and demonstrated that the choice of carbon sources did not significantly change the synthesis and activity of the enzyme. The DNA sequence of the Streptomyces lividans glucose kinase gene glkA was determined. The predicted gene product of 317 amino acids was found to be identical to S. coelicolor glucose kinase, suggesting a similar role for this protein in both organisms. A procedure was developed to produce pure histidine-tagged glucose kinase with a yield of approximately 10 mg/l culture. The protein was stable for several weeks and was used to raise polyclonal antibodies. Purified glucose kinase was used to explore protein-protein interaction by surface plasmon resonance. The experiments revealed the existence of a binding activity present in S. coelicolor cell extracts. This indicated that glucose kinase may interact with (an)other factor(s), most likely of protein nature. A possible cross-talk with proteins of the phosphotransferase system, which are involved in carbon catabolite repression in other bacteria, was investigated.     

Genomic structure and fine mapping of the two human filamin gene paralogues FLNB and FLNC and comparative analysis of the filamin gene family

The genomic structure of the filamin gene paralogues FLNB and FLNC was determined and related to FLNA. FLNB consists of 45 exons and 44 introns and spans approximately 80 kb of genomic DNA. FLNC is divided into 48 exons and 47 introns and covers approximately 29.5 kb of genomic DNA. A previously unknown intron was found in FLNA. The comparison of all three filamin gene paralogues revealed a highly conserved exon-intron structure with significant differences in the exons 32 of all paralogues encoding the hinge I region, as well as the insertion of a novel exon 40A in FLNC only. Gene organization does not correlate with the domain structures of the respective proteins. To improve candidate gene cloning approaches, FLNB was precisely mapped at 3p14 in an interval of 0.81 cM between WI3771 and WI6691 and FLNC at 7q32 in an interval of 2.07 cM between D7S530 and D7S649.     

Radial transport of abscisic acid conjugates in maize roots: its implication for long distance stress signals

Conjugated, alkaline hydrolysable ABA (predominantly abscisic acid glucose ester, ABA-GE), which is transported in the xylem from roots to shoots of Zea mays L. plants, has its origin in the root symplast rather than from soil, although it was detectable in soil solution with concentrations up to 30 nM. External ABA glucose ester cannot be dragged with the water flow across the exodermis and the endodermis because of its hydrophobic properties. Experimental evidence is presented that enzymes in the cortical apoplast cleave ABA-GE thus releasing ABA from its conjugates. Liberated ABA can then be translocated apoplastically and symplastically to the xylem vessels. Endogenous ABA-GE can be released from isolated cortical and stelar tissues to the surrounding media, with rates that are up to 5-fold higher from stelar tissues than those from cortical tissues. Release of ABA-GE is highest under conditions of inhibited ABA-metabolism.     

A rev-like NES mediates cytoplasmic localization of HERV-K cORF

The human endogenous retrovirus K (HERV-K)-encoded protein cORF has recently been shown to be a functional homolog of the HIV Rev protein. Rev-mediated RNA export requires interaction between a leucine-rich nuclear export signal (NES) in Rev and the nuclear export receptor Crm1/exportin1. Like Rev, cORF binds to Crm1 and cORF-mediated RNA export depends on Crm1 activity. Here we document that mutation of the putative NES in cORF results in trapping of the protein in the nucleus, suggesting that the cORF NES functions in analogy to the Rev NES.