Human RFP2 gene promoter: unique structure and unusual strength

Human gene RFP2 is a candidate tumor suppressor located at 13q14.3 and deleted in multiple tumor types. To explore regulation of RFP2, we determined structure of the 5'-untranslated region of RFP2 gene and its promoter. RFP2 promoter area is TATA-less, highly enriched in G and C nucleotides, and contains multiple quadruplex forming GGGGA-repeats. Deletion analysis of 5'-flanking sequences demonstrated that repeat containing fragment possesses activity seven times exceeding that of the combined SV40 promoter/enhancer. Other unusual features of the RFP2 promoter include anomalously high electrostatic fields induced by sequence-dependent dipoles and very low nucleosome forming potential. A "minimized" version of the RFP2 promoter could be used for overexpression of the various transgenes in the mammalian cells.     

A new automated technique for the reconstitution of hydrophobic proteins into planar bilayer membranes. Studies of human recombinant uncoupling protein 1

Electrophysiological characterisation of the vast number of annotated channel and transport proteins in the postgenomic era would be greatly facilitated by the introduction of rapid and robust methods for the functional incorporation of membrane proteins into defined lipid bilayers. Here, we describe an automated technique for reconstitution of membrane proteins into lipid bilayer membranes, which substantially reduces both the reconstitution time and the amount of protein required for the membrane formation. The method allows the investigation of single protein channels as well as insertion of multiple copies (approximately 10(7)) into a single bilayer. Despite a comparatively large membrane area (up to 300 microm diameter), the high stability of the membrane permits the application of transmembrane voltages up to 300 mV. This feature is especially important for studies of inner membrane mitochondrial proteins, since they act at potentials up to approximately 200 mV under physiological conditions. It is a combination of these advantages that enables the detailed investigation of the minuscule single protein conductances typical for proton transporters. We have applied the new technique for the reconstitution and electrophysiological characterisation of human recombinant uncoupling protein 1, hUCP1, that has been overexpressed in E. coli and purified from inclusion bodies. We demonstrate that hUCP1 activity in the presence of fatty acids is comparable to the activity of UCP1 isolated from brown adipose tissue.     

Involvement of Y-family DNA polymerases in mutagenesis caused by oxidized nucleotides in Escherichia coli

Escherichia coli DNA polymerase IV incorporated 2-hydroxy-dATP opposite template guanine or thymine and 8-hydroxy-dGTP exclusively opposite adenine in vitro. Mutator phenotypes in sod/fur strains were substantially diminished by deletion of dinB and/or umuDC. DNA polymerases IV and V may be involved in mutagenesis caused by incorporation of the oxidized deoxynucleoside triphosphates.     

Upregulation of jasmonate-inducible defense proteins and differential colonization of roots of Oryza sativa cultivars with the endophyte Azoarcus sp

The endophyte Azoarcus sp. strain BH72 expresses nitrogenase (nif) genes inside rice roots. We applied a proteomic approach to dissect responses of rice roots toward bacterial colonization and jasmonic acid (JA) treatment. Two sister lineages of Oryza sativa were analyzed with cv. IR42 showing a less compatible interaction with the Azoarcus sp. resulting in slight root browning whereas cv. IR36 was successfully colonized as determined by nifHi::gusA activity. External addition of JA inhibited colonization of roots and caused browning in contrast to the addition of ethylene, applied as ethephon (up to 5 mM). Only two of the proteins induced in cv. IR36 by JA were also induced by the endophyte (SalT, two isoforms). In contrast, seven JA-induced proteins were also induced by bacteria in cv. IR42, indicating that IR42 showed a stronger defense response. Mass spectrometry analysis identified these proteins as pathogenesis-related (PR) proteins (Prb1, RSOsPR10) or proteins sharing domains with receptorlike kinases induced by pathogens. Proteins strongly induced in roots in both varieties by JA were identified as Bowman-Birk trypsin inhibittors, germinlike protein, putative endo-1,3-beta-D-glucosidase, glutathion-S-transferase, and 1-propane-1-carboxylate oxidase synthase, peroxidase precursor, PR10-a, and a RAN protein previously not found to be JA-induced. Data suggest that plant defense responses involving JA may contribute to restricting endophytic colonization in grasses. Remarkably, in a compatible interaction with endophytes, JA-inducible stress or defense responses are apparently not important.     

A heterogeneous immunofluorescence staining for laminin-1 and related basal lamina molecules in the dorsal root ganglia following constriction nerve injury

The bodies of primary sensory neurons and their satellite glial cells (SGCs) are limited by the basal laminae from extracellular matrix of the dorsal root ganglia (DRG). The basal laminae displayed uniform immunofluorescence staining for laminin-1 in the sections of rat intact (naive) DRG. A proximal or distal ligature of the spinal nerves resulted in a heterogeneous immunostaining for laminin-1 around neuron-SGC units in the sections of the corresponding DRG. The pattern of irregular laminin-1 immunofluorescence was more extensive in the ipsilateral than the contralateral DRG of the operated rats. The immunofluorescence for laminin-1 exactly coincided with binding of Concanavalin-A as well as immunostaining for type IV collagen in both naive DRG and DRG affected by nerve ligature. Nidogen immunostaining decreased or fully disappeared at the surface of the SGCs consistently with immunofluorescence staining for laminin-1, but retained or increased in the endothelial cells and ED-1 positive cells invaded the DRG affected by nerve ligature. The results indicate an alteration of the content of basal laminae surrounding the bodies of primary sensory neurons and their SGSs following nerve constriction injury. A modulation of the basal laminae may be related with other cellular and molecular alterations related with peripheral neuropathic pain, for example, expansion of sympathetic sprouts.     

Development of a combined setup for simultaneous detection of total and glycated haemoglobin content in blood samples

An original setup for analysis of glycated haemoglobin (HbA1c) in blood is reported. The construction employed a combination of the piezoelectric biosensor for glycated haemoglobin and the flow-through photometric sensor for total haemoglobin (Hb). The modification of gold electrodes with 3-aminophenylboronic acid (APBA) as a specific ligand was studied; the chemisorbed conjugate of APBA with a long-chain thiocompound provided the best affinity for HbA1c. The effect of various operating parameters, such as flow rate and instrumental setup, was optimised. The total haemoglobin content was analysed as absorbance of the haemoglobin-cyanide derivative at 540 nm. Only one standard (calibrator) diluted in various ratio was necessary for calibration and 1 microl of blood was sufficient for analysis. The full range of HbA1c content (4-15%) in blood can be analysed; the working ranges of total and glycated haemoglobin were 50-2000 and 10-90 microg/ml, respectively. The developed method was successfully evaluated on blood samples collected from diabetics.     

Suzuki-type Pd(0) coupling reactions in the synthesis of 2-arylpurines as Cdk inhibitors

A new series of 2-aryl-substituted purine derivatives has been synthesized by Suzuki Pd(0) coupling reactions. Moderate in vitro inhibitory activity against Cdk1 and Cdk5 was observed. These compounds are inactive against GSK3.     

The tick plasma lectin, Dorin M, is a fibrinogen-related molecule

A lectin, named Dorin M, previously isolated and characterized from the hemolymph plasma of the soft tick, Ornithodoros moubata, was cloned and sequenced. The immunofluorescence using confocal microscopy revealed that Dorin M is produced in the tick hemocytes. A tryptic cleavage of Dorin M was performed and the resulting peptide fragments were sequenced by Edman degradation and/or mass spectrometry. Two of three internal peptide sequences displayed a significant similarity to the family of fibrinogen-related molecules. Degenerate primers were designed and used for PCR with hemocyte cDNA as a template. The sequence of the whole Dorin M cDNA was completed by the method of RACE. The tissue-specific expression investigated by RT-PCR revealed that Dorin M, in addition to hemocytes, is significantly expressed in salivary glands. The derived amino-acid sequence clearly shows that Dorin M has a fibrinogen-like domain, and exhibited the most significant similarity with tachylectins 5A and 5B from a horseshoe crab, Tachypleus tridentatus. In addition, other protein and binding characteristics suggest that Dorin M is closely related to tachylectins-5. Since these lectins have been reported to function as non-self recognizing molecules, we believe that Dorin M may play a similar role in an innate immunity of the tick and, possibly, also in pathogen transmission by this vector.     

Biological activities as patchiness driving forces in wetlands of northern Belize

Patchiness in wetlands is a common and well documented phenomenon. Oligotrophic wetlands of northern Belize display noticeable vegetation heterogeneity at both large and small scales. In this paper, we document the small scale patches in herbaceous wetlands, describe differences between patches and surrounding wetland habitats and explain patch formation and sustenance. We conducted a survey of patches and confirmed their occurrence by spatial analysis. Patches were distinguished from a surrounding wetland by denser and taller vegetation, higher amount of empty snail shells and elevated soil phosphorus (P). Plants in patches had higher tissue nitrogen (N) and P content and there was also higher total N and P per m² incorporated in plant biomass. In terms of stable isotopes, plants in patches were enriched in ¹⁵N; patch soils were depleted in ¹³C. Observations of focal individuals of Aramus guarauna, limpkin, a wading bird feeding almost exclusively on snails, revealed the origin of the snail shell piles frequently found in patches. An adult limpkin captured on average 18 snails daily, of these 80% were handled in patches and birds often repeatedly used the same patch. Experimental patch creation by adding chicken manure or P to 1 m² plots resulted in higher and denser vegetation with values increasing in order: control, P, manure plots. The effect was significant at both experimental locations six months after the treatment and at one location even 40 months after the treatment. We present a simple mechanistic explanation for nutrient redistribution in wetlands and their eventual accumulation in patches. Both nutrient and isotopic differences result from animal input into patches, e.g. bird droppings or prey remnants. Foraging activity of Aramus guarauna is most likely responsible for patch formation. A positive feedback (repeated use of a suitable patch) is apparently the mechanism sustaining patches in these marsh environments.