Anterior repression of a Drosophila stripe enhancer requires three position-specific mechanisms

The striped expression pattern of the pair-rule gene even skipped (eve) is established by five stripe-specific enhancers, each of which responds in a unique way to gradients of positional information in the early Drosophila embryo. The enhancer for eve stripe 2 (eve 2) is directly activated by the morphogens Bicoid (Bcd) and Hunchback (Hb). As these proteins are distributed throughout the anterior half of the embryo, formation of a single stripe requires that enhancer activation is prevented in all nuclei anterior to the stripe 2 position. The gap gene giant (gt) is involved in a repression mechanism that sets the anterior stripe border, but genetic removal of gt (or deletion of Gt-binding sites) causes stripe expansion only in the anterior subregion that lies adjacent to the stripe border. We identify a well-conserved sequence repeat, (GTTT)(4), which is required for repression in a more anterior subregion. This site is bound specifically by Sloppy-paired 1 (Slp1), which is expressed in a gap gene-like anterior domain. Ectopic Slp1 activity is sufficient for repression of stripe 2 of the endogenous eve gene, but is not required, suggesting that it is redundant with other anterior factors. Further genetic analysis suggests that the (GTTT)(4)-mediated mechanism is independent of the Gt-mediated mechanism that sets the anterior stripe border, and suggests that a third mechanism, downregulation of Bcd activity by Torso, prevents activation near the anterior tip. Thus, three distinct mechanisms are required for anterior repression of a single eve enhancer, each in a specific position. Ectopic Slp1 also represses eve stripes 1 and 3 to varying degrees, and the eve 1 and eve 3+7 enhancers each contain GTTT repeats similar to the site in the eve 2 enhancer. These results suggest a common mechanism for preventing anterior activation of three different eve enhancers.     

Digital glomus tumors: a 29-year experience with a lateral subperiosteal approach

The excision of distal digital glomus tumors has traditionally been performed directly over the involved nail bed. This can lead to nail deformities that are often unacceptable for the surgeon and the patient. The authors describe their experience with successful excision of digital glomus tumors using a lateral subperiosteal approach, which creates a dorsal flap. In 29 years, 19 patients were diagnosed with digital glomus tumors. All patients underwent excision using the lateral subperiosteal approach. The mean tumor size was 0.52 cm. The tumors were located on the pulp of the distal phalanx in two patients (10.5 percent) and subungually in 17 patients (89.5 percent). In all patients, preoperative clinical diagnosis was confirmed postoperatively with the biopsy result. Complications occurred in only two patients and included one paronychia and one temporary nail loss. The overall recurrence rate was 15.7 percent. All patients remained asymptomatic after surgery and regained full active and passive range of motion. There were no nail deformities by this approach. This technique represents a safe and effective approach to excising digital glomus tumors.     

The crystal structure of the globular domain of sheep prion protein

The prion protein PrP is a naturally occurring polypeptide that becomes transformed from a normal conformation to that of an aggregated form, characteristic of pathological states in fatal transmissible spongiform conditions such as Creutzfeld-Jacob Disease and Bovine Spongiform Encephalopathy. We report the crystal structure, at 2 A resolution, of residues 123-230 of the C-terminal globular domain of the ARQ allele of sheep prion protein (PrP). The asymmetric unit contains a single molecule whose secondary structure and overall organisation correspond to those structures of PrPs from various mammalian species determined by NMR. The globular domain shows a close association of helix-1, the C-terminal portion of helix-2 and the N-terminal portion of helix-3, bounded by the intramolecular disulphide bond, 179-214. The loop 164-177, between beta2 and helix-2 is relatively well structured compared to the human PrP NMR structure. Analysis of the sheep PrP structure identifies two possible loci for the initiation of beta-sheet mediated polymerisation. One of these comprises the beta-strand, residues 129-131 that forms an intra-molecular beta-sheet with residues 161-163. This strand is involved in lattice contacts about a crystal dyad to generate a four-stranded intermolecular beta-sheet between neighbouring molecules. The second locus involves the region 188-204, which modelling suggests is able to undergo a partial alpha-->beta switch within the monomer. These loci provide sites within the PrPc monomer that could readily give rise to early intermediate species on the pathway to the formation of aggregated PrPSc containing additional intermolecular beta-structure.     

Salt sensitivity of minimal twin arginine translocases

Bacterial twin arginine translocation (Tat) pathways have evolved to facilitate transport of folded proteins across membranes. Gram-negative bacteria contain a TatABC translocase composed of three subunits named TatA, TatB, and TatC. In contrast, the Tat translocases of most Gram-positive bacteria consist of only TatA and TatC subunits. In these minimal TatAC translocases, a bifunctional TatA subunit fulfils the roles of both TatA and TatB. Here we have probed the importance of conserved residues in the bifunctional TatAy subunit of Bacillus subtilis by site-specific mutagenesis. A set of engineered TatAy proteins with mutations in the cytoplasmic hinge and amphipathic helix regions were found to be inactive in protein translocation under standard growth conditions for B. subtilis or when heterologously expressed in Escherichia coli. Nevertheless, these mutated TatAy proteins did assemble into TatAy and TatAyCy complexes, and they facilitated membrane association of twin arginine precursor proteins in E. coli. Interestingly, most of the mutated TatAyCy translocases were salt-sensitive in B. subtilis. Similarly, the TatAC translocases of Bacillus cereus and Staphylococcus aureus were salt-sensitive when expressed in B. subtilis. Taken together, our present observations imply that salt-sensitive electrostatic interactions have critical roles in the preprotein translocation activity of certain TatAC type translocases from Gram-positive bacteria.     

Association of polymorphism in the thermolabile 5, 10-methylene tetrahydrofolate reductase gene and hyperhomocysteinemia with coronary artery disease

Objective To determine the incidence of methylene tetrahydrofolate reductase (MTHFR) gene 677C→T polymorphism and plasma homocysteine (Hcy) levels in a group of subjects who underwent coronary angiography, in an attempt to establish a correlation between these parameters and the severity of coronary artery disease (CAD) and to investigate the correlation between hyperhomocysteinemia (HHcy) and the presence of 677C→T polymorphism. Background Elevated plasma Hcy level is an independent risk factor for CAD. A common mutation (677C→T) in the gene coding for MTHFR has been reported to reduce the enzymatic activity and is associated with elevated levels of Hcy, especially in subjects with low folate intake. Methods The study group comprised of 84 patients with CAD and 100 age-and-sex matched controls who had no history or clinical evidence of CAD and/or MI. DNA was extracted from peripheral blood and genotypes were determined by polymerase chain reaction, restriction mapping with Hinf1, and gel electrophoresis. Conventional risk factors for CAD were prospectively documented. Results Allele and genotype frequencies in cases and control subjects were compatible with Hardy–Weinberg equilibrium. The frequencies of TT, CT, and CC genotypes among CAD patients were 4.8, 27.4, and 67.8% and in controls were 1.0, 19.0, and 80%. Hcy levels were higher in patients with triple-vessel disease compared to single and double vessel disease (P = 0.002). Multivariate analyses identified HHcy, diabetes mellitus, and hypertension as the independent predictors of CAD. Conclusions HHcy appears to have a graded effect on the risk of CAD as well as the severity and extent of coronary atherosclerosis. Our findings support that homozygous genotype of MTHFR is a genetic risk factor for CAD. A further study with larger sample size including assessment of vitamin status is needed to better clarify the relationship between MTHFR genotypes and CAD.     

The effects of stem I and loop A on the processing of 5 S rRNA from Drosophila melanogaster.

The 135-nucleotide Drosophila melanogaster 5 S RNA precursor is processed by removal of 15 nucleotides from its 3' end before incorporation into the large ribosomal subunit. Mature 5 S RNA consists of five helical stem-loops; stem IV and part of V are dispensable, whereas stem III and the 1/118 G-C base pair closest to the processing site at nucleotide 120 are required for processing (Preiser, P., and Levinger, L. (1991) J. Biol. Chem. 266, 7509-7516; Preiser, P., and Levinger, L. (1991) J. Biol. Chem. 266, 23602-23605). We have investigated the effects of stem I and loop A transversions, transitions, selected additions and deletions on 5 S RNA processing. Stem I single substitutions generally prevent processing, whereas compensatory double substitutions restore a range of processing rates. Proximal to the processing site, stem I double substitutions inhibit processing. In the distal portion of stem I and loop A, the processing effect of paired sequence changes varies widely in an irregular pattern. The 7/112 GU pair and nucleotide 13A least tolerate sequence changes; several mutations clustered close to the stem I-loop A boundary stimulate processing. We interpret these results in terms of the RNA helix path and possible RNA-protein contacts.