Interactions of the nuclear matrix-associated steroid receptor binding factor with its DNA binding element in the c-myc gene promoter

Steroid receptor binding factor (RBF) was originally isolated from avian oviduct nuclear matrix. When bound to avian genomic DNA, RBF generates saturable high-affinity binding sites for the avian progesterone receptor (PR). Recent studies have shown that RBF binds to a 54 bp element in the 5'-flanking region of the progesterone-regulated avian c-myc gene, and nuclear matrix-like attachment sites flank the RBF element [Lauber et al. (1997) J. Biol. Chem. 272, 24657-24665]. In this paper, electrophoretic mobility shift assays (EMSAs) and S1 nuclease treatment are used to demonstrate that the RBF-maltose binding protei (MBP) fusion protein binds to single-stranded DNA of its element. Only the N-terminal domain of RBF binds the RBF DNA element as demonstrated by southwestern blot analyses, and by competition EMSAs between RBF-MBP and the N-terminal domain. Mass spectrometric analysis of the C-terminal domain of RBF demonstrates its potential to form noncovalent protein-protein interactions via a potential leucine-isoleucine zipperlike structure, suggesting a homo- and/or possible heterodimer structure in solution. These data support that the nuclear matrix binding site (acceptor site) for PR in the c-myc gene promoter is composed of RBF dimers bound to a specific single-stranded DNA element. The dimers of RBF are generated by C-terminal leucine zipper and the DNA binding occurs at the N-terminal parallel beta-sheet DNA binding motif. This complex is flanked by nuclear matrix attachment sites.     

Modulation of N-type calcium channel activity by G-proteins and protein kinase C

N-type voltage-gated calcium channel activity in rat superior cervical ganglion neurons is modulated by a variety of pathways. Activation of heterotrimeric G-proteins reduces whole-cell current amplitude, whereas phosphorylation by protein kinase C leads to an increase in current amplitude. It has been proposed that these two distinct pathways converge on the channel's pore-forming alpha(1B) subunit, such that the actions of one pathway can preclude those of the other. In this study, we have characterized further the actions of PKC on whole-cell barium currents in neonatal rat superior cervical ganglion neurons. We first examined whether the effects of G-protein-mediated inhibition and phosphorylation by PKC are mutually exclusive. G-proteins were activated by including 0.4 mM GTP or 0.1 mM GTP-gamma-S in the pipette, and PKC was activated by bath application of 500 nM phorbol 12-myristate 13-acetate (PMA). We found that activated PKC was unable to reverse GTP-gamma-S-induced inhibition unless prepulses were applied, indicating that reversal of inhibition by phosphorylation appears to occur only after dissociation of the G-protein from the channel. Once inhibition was relieved, activation of PKC was sufficient to prevent reinhibition of current by G-proteins, indicating that under phosphorylating conditions, channels are resistant to G-protein-mediated modulation. We then examined what effect, if any, phosphorylation by PKC has on N-type barium currents beyond antagonizing G-protein-mediated inhibition. We found that, although G-protein activation significantly affected peak current amplitude, fast inactivation, holding-potential-dependent inactivation, and voltage-dependent activation, when G-protein activation was minimized by dialysis of the cytoplasm with 0.1 mM GDP-beta-S, these parameters were not affected by bath application of PMA. These results indicate that, under our recording conditions, phosphorylation by PKC has no effect on whole-cell N-type currents, other than preventing inhibition by G-proteins.     

MEROPS: the peptidase database

Important additions have been made to the MEROPS database (http://www.bi.bbsrc.ac.uk/Merops/Merops.htm). These include sequence alignments and cladograms for many of the families of peptidases, and these have proved very helpful in the difficult task of distinguishing the sequences of peptidases that are simply species variants of already known enzymes from those that represent novel enzymes.     

Sodium intake is increased by social stress and the Y chromosome and reduced by clonidine

The objectives were to determine 1) if female rats have higher Na intake than males and if social stress increases Na intake, 2) if the sympathetic nervous system (SNS) mediates the stress effects and the gender effect, and 3) if the Y chromosome (Yc) from a hypertensive father increases Na intake. Four rat strains (n = 10/group) of both sexes were used: 1) Wistar Kyoto normotensive (WKY), 2) an F(16) backcross with a Yc from a hypertensive father (SHR/y), 3) spontaneously hypertensive rat (SHR), and 4) an F(16) backcross with a Yc from a normotensive father (SHR/a). Females showed greater baseline Na intake than males (hypertensive strains), intruder stress increased Na intake, and clonidine decreased Na intake, but not in WKY or SHR females. SHR/y males had higher baseline Na intake compared with WKY males. In conclusion, the higher Na intake in females during baseline and stress was partially mediated through the SNS in hypertensive strains and the SHR Yc was partially responsible for the increased Na intake in SHR/y and SHR males compared with WKY.     

Cloning,sequencing and analysis of the pucC genes from Rubrivivax gelatinosus strain 151 and Rhodopseudomonas acidophila strain 10050

The pucC genes of Rubrivivax gelatinosus strain 151 and Rhodopseudomonas acidophila strain 10050 have been identified, cloned and sequenced. In Rubrivivax gelatinosus the arrangement of the pucC gene with regard to the pucBA genes was shown to differ from that found in other species of photosynthetic bacteria. The Rhodopseudomonas acidophila pucC was found downstream of four new pucBA gene pairs, bringing the sequenced pucBA pairs to a total of eight in this strain. The predicted PucC protein sequences were compared to those of PucC from other species and showed high similarity. Similarity was also seen to more distantly related proteins LhaA and orf428 of Rhodobacter capsulatus, orf G115 of Rhodospirillum rubrum and `orf428' from Synechocystis sp. PCC6803. An analysis of the predicted secondary structure of these proteins is given, and their structural similarity to proteins in the Major Facilitator Superfamily is discussed with regard to their possible function. This revised version was published online in June 2006 with corrections to the Cover Date.     

Realities of diagnosing Helicobacter pylori infection in clinical practice: a case for non-invasive indirect methodologies.

BACKGROUND: The current, arbitrarily defined gold standard for the diagnosis of H. pylori infection requires histologic examination of two specially stained antral biopsy specimens. However, routine histology is potentially limited in general clinical practice by both sampling and observer error. The current study was designed to examine the diagnostic performance of invasive and non-invasive H. pylori detection methods that would likely be available in general clinical practice. METHODS: The diagnostic performance of rotating clinical pathology faculty using thiazine staining was compared with that of an expert gastrointestinal pathologist in 38 patients. In situ hybridization stains of adjacent biopsy cuts were also examined by the expert pathologist for further comparison. Receiver operator characteristic (ROC) analysis was performed to evaluate whether the diagnostic performance of the expert pathologist differed depending upon the histologic method employed. A similar analysis was made to evaluate the diagnostic performance of pathology trainees relative to the expert. In the absence of an established invasive gold standard, non-invasive testing methods (rapid serum antibodies, formal Elisa antibodies and carbon-14 urea breath testing) were evaluated in 74 patients by comparison with a gold standard defined using a combination of diagnostic tests. RESULTS: Using either rapid urease testing of biopsy specimens or urea breath testing as the gold standard for comparison, the diagnostic performance of the rotating clinical pathology faculty was inferior to that of the expert gastrointestinal pathologist especially with regard to specificity (e.g., 69 percent for the former versus 88 percent, with the latter relative to rapid urease testing). Although interpretation of in situ hybridization staining by the expert appeared to have an even higher specificity, ROC analysis failed to show a difference. The mean ROC areas for thiazine and in situ hybridization staining for trainee pathologists relative to the expert were 0.88 and 0.94, respectively. In untreated patients, urea breath testing had a sensitivity and specificity of 100 percent as compared with thiazine staining with a sensitivity of 83 percent and a specificity of 97 percent. Post-therapy, breath testing had a sensitivity of 100 percent but a specificity of only 86 percent as compared with invasive testing with a sensitivity and specificity of 100 percent. Rapid serum antibody testing and formal Elisa antibody testing agreed in 93 percent of cases (Kappa 0.78) with the rapid test being correct in three of the four disagreements. CONCLUSIONS: The current study illustrates a number of realities regarding H. pylori diagnosis. There is no diagnostic gold standard in general clinical practice. Accurate interpretation of specially stained slides is a learned activity with a tendency towards overdiagnosis early on. Urea breath testing is likely to be the diagnostic method of choice for untreated patients in general clinical practice although antibody testing is almost as accurate. Rapid antibody tests are at least as accurate as formal Elisa antibody tests. Urea breath testing is useful for confirming cure after therapy, but false-positive results may occur in some patients.     

Application of conformationally restricted peptidomimetics to modeling the bound conformation of peptide antagonists with the IL-1 receptor

A collection of complementary peptide caricatures that closely mimic low-energy (presumably highly populated) conformations of amino acids of interest would constitute a valuable tool set to study the interactions of small peptide ligands with their biological targets. Our general strategy for the design, synthesis and application of peptidomimetics is presented. An illustration of how structural information from mimetics combined with cutting edge biophysical data can be used to derive a model for the bound conformation of an 11-mer peptide antagonist with the IL-1 receptor is given.     

On Hybrid Formation in the Rock Fern Asplenium x alternifolium (Aspleniaceae,Pteridophyta)

Length polymorphism in a non-coding spacer (trnL_UAA-trnF_GAA) in the chloroplast DNA was used in the investigation of the origin of the most common and conspicuous European fern hybrid, Asplenium x alternifolium (Aspleniaceae, Pteridophyta). The origins of A. x alternifolium, the hybrid between A. trichomanes s.l. and A. septentrionale s.l. was studied at three ploidy levels, diploid, triploid and tetraploid. The cpDNA technique allowed us to investigate the mode of hybrid formation between sexual species for the first time over a wide geographic range and with a large sample size. Morphological variation in this hybrid has previously been attributed to different reciprocal parental combinations, and to the influence of chloroplast genes on morphogenesis. Our results demonstrate that one parent, A. septentrionale s.l., acts predominantly as the female parent in these hybrids, with only one population of A. x alternifolium showing reciprocal hybridity. The discovery of predominantly unidirectional hybrid formation in this hybrid may be explained by the different breeding systems of the parental taxa. The role of gametophyte ecology is also assessed.     

Climate,Colonisation and Celibacy: Population Structure in Central European Trichomanes speciosum (Pteridophyta)

The Killarney fern Trichomanes speciosum Willd. (Hymenophyllaceae) is unique in possessing both extensive sexual (sporophyte and gametophyte generations present) and asexual (gametophyte only) ranges. It was first discovered in central Europe in 1993 and is represented in this area only by its perennial, vegetatively propagating gametophyte generation. Genetic variation has been investigated at 35 sites. Allozyme diversity is partitioned primarily between, not within, sites. Although genetic variation exists at a fine scale (lt 5 m) within some populations, the results suggest that clones were not intimately associated in these cases. The majority of sites support unique multilocus phenotypes. Where phenotypes were present at more than one site they tended to recur at the next closest site. However, similar phenotypes link eastern and western Pfälzerwald sites up to c. 70 km apart. This pattern of diversity suggests that colonisation was not solely of a “stepping stone” or “leading edge” type. We suggest that during a climatically favourable period, probably the Atlantic hypsithermal, there may have been an explosive colonisation by long-distance dispersal from refugial areas. This was followed by a short period during which sporophyte production, sexual reproduction and local spread were possible. With climatic change, reduction in the available habitat and the loss of the sporophyte generation, different individual genets became fixed within small, favourable, but scattered, sites. The possibility that some central European sites north of the Alps acted as periglacial refugia cannot be discounted, but would appear less likely than (re-) colonisation from the Atlantic fringe.