Solution conformations of unmodified and A(37)N(6)-dimethylallyl modified anticodon stem-loops of Escherichia coli tRNA(Phe)

The modification of RNA nucleotide bases, a fundamental process in all cells, alters the chemical and physical properties of RNA molecules and broadly impacts the physiological properties of cells. tRNA molecules are by far the most diverse-modified RNA species within cells, containing as a group >80% of the known 96 chemically unique nucleic acid modifications. The greatest varieties of modifications are located on residue 37 and play a role in ensuring fidelity and efficiency of protein synthesis. The enzyme dimethylallyl (Delta(2)-isopentenyl) diphosphate:tRNA transferase catalyzes the addition of a dimethylallyl group to the exocyclic amine nitrogen (N6) of A(37) in several tRNA species. Using a 17 residue oligoribonucleotide corresponding to the anticodon arm of Escherichia coli tRNA(Phe), we have investigated the structural and dynamic changes introduced by the dimethylallyl group. The unmodified RNA molecule adopts stem-loop conformation composed of seven base-pairs and a compact three nucleotide loop. This conformation is distinctly different from the U-turn motif that characterizes the anticodon arm in the X-ray crystal structure of the fully modified yeast tRNA(Phe). The adoption of the tri-nucleotide loop by the purine-rich unmodified tRNA(Phe) anticodon arm suggests that other anticodon sequences, especially those containing pyrimidine bases, also may favor a tri-loop conformation. Introduction of the dimethylallyl modification increases the mobility of nucleotides of the loop region but does not dramatically alter the RNA conformation. The dimethylallyl modification may enhance ribosome binding through multiple mechanisms including destabilization of the closed anticodon loop and stabilization of the codon-anticodon helix.     

Properties of the glucose-6-phosphate transporter from Chlamydia pneumoniae (HPTcp) and the glucose-6-phosphate sensor from Escherichia coli (UhpC)

The amino acid sequence of the proposed glucose-6-phosphate (Glc6P) transporter from Chlamydia pneumoniae (HPTcp; hexose phosphate transporter [Chlamydia pneumoniae]) exhibits a higher degree of similarity to the Escherichia coli Glc6P sensor (UhpC) than to the E. coli Glc6P transporter (UhpT). Overexpression of His-UhpC in a UhpT-deficient E. coli strain revealed that the sensor protein is also able to transport Glc6P and exhibits an apparent K(m) ((Glc6P)) of 25 microM, whereas His-HPTcp exhibits an apparent K(m)( (Glc6P)) of 98 microM. His-HPTcp showed a four-times-lower specific activity than His-UhpT but a 56-times-higher specific activity than His-UhpC. Like His-UhpT and His-UhpC, the carrier His-HPTcp performs a sugar-phosphate/inorganic-phosphate antiporter mode of transport. Surprisingly, while physiological concentrations of inorganic phosphate competitively inhibited transport mediated by the E. coli proteins His-UhpT and His-UhpC, transport mediated by His-HPTcp was not inhibited. Interestingly, C(3)-organophosphates stimulated His-HPTcp activity but not His-UhpT- or His-UhpC-catalyzed Glc6P transport. In contrast to His-UhpC, the His-HPTcp protein does not act as a Glc6P sensor in the uhp regulon.     

Topical application of dynorphin A (1-17) antiserum attenuates trauma induced alterations in spinal cord evoked potentials,microvascular permeability disturbances,edema formation and cell injury: an experimental study in the rat using electrophysiological and morphological approaches

Summary.  Dynorphin is a neuropeptide that is present in high quantities in the dorsal horn of the spinal cord. The peptide is actively involved in pain processing pathways. However, its involvement in spinal cord injury is not well known. Alteration in dynorphin immunoreactivity occurs following a focal trauma to the rat spinal cord. Infusion of dynorphin into the intrathecal space of the cord results in ischemia, cell damage and abnormal motor function. Antibodies to dynorphin when injected into the intrathecal space of the spinal cord following trauma improve motor recovery, reduce edema and cell changes. However, influence of dynorphin on trauma induced alteration in spinal cord bioelectrical activity is still not known. Spinal cord evoked potentials (SCEP) are good indicator of spinal cord pathology following trauma. Therefore, in present investigation, influence of dynorphin antibodies on trauma induced changes in SCEP were examined in our rat model. In addition, spinal cord edema formation, microvascular permeability disturbances and cell injury were also investigated. Our results show that topical application of dynorphin antiserum (1 : 200) two min before injury markedly attenuated the SCEP changes immediately after injury. In the antiserum treated animals, a significant reduction in the microvascular permeability, edema formation and cell injury was observed in the traumatised spinal cord. These observations suggest that (i) dynorphin is involved in the altered bioelectrical activity of the spinal cord following trauma, (ii) the peptide actively participates in the pathophysiological processes of cell injury in the spinal cord trauma, and (iii) the dynorphin antiserum has potential therapeutic value for the treatment of spinal cord injuries. Received July 3, 2001 Accepted August 6, 2001 Published online July 31, 2002     

Preweaning enrichment has no lasting effects on adult hippocampal neurogenesis in four-month-old mice

Since both living in an enriched environment and physical activity stimulate hippocampal neurogenesis in adult mice, we endeavored to examine whether preweaning enrichment, a sensory enrichment paradigm with very limited physical activity, had similar effects on neurogenesis later in life. Mice were removed from the dams for periods of increasing length from postnatal day 7 to 21, and exposed to a variety of sensory stimuli. At the age of 4 months, significant differences could be found between previously enriched and non-enriched animals when spontaneous activity was monitored. Enriched mice moved longer distances, and spent more time in a defined center zone of the open field. Adult neurogenesis was examined by labeling proliferating cells in the dentate gyrus with bromodeoxyuridine (BrdU). Cell proliferation, survival of the newborn cells, and net neurogenesis were similar in both groups. Volumetric measurements and stereological assessment of total granule cell counts revealed no difference in size of the dentate gyrus between both groups. Thus, in contrast to postweaning enrichment, preweaning enrichment had no lasting measurable effect on adult neurogenesis. One of the parameters responsible for this effect might be the lack of physical activity in preweaning enrichment. As physical activity is an integral part of postweaning enrichment, it might be a necessary factor to elicit a neurogenic response to environmental stimuli. The result could also imply that baseline adult hippocampal neurogenesis is independent of the changes induced by preweaning enrichment and might not contribute to the sustained types of plasticity seen in enriched animals.     

Development of a 1-microl scale assay for mitogen-activated kinase kinase 7 using 2-D fluorescence intensity distribution analysis anisotropy

This paper describes the development of a robust, miniaturizable, and quantitative fluorescence-based assay for mitogen-activated protein kinase kinase 7 (MKK7). As a first step, the basic steady-state kinetics of the MKK7-catalyzed phosphorylation of c-Jun N-terminal kinases (JNKs) 1, 2, and 3 were defined using standard radiometric methods. Subsequently, the authors found that in addition to the holo JNKs, a series of novel small peptides (based on the region around the JNK phosphorylation site) are also substrates, provided that these were prephosphorylated on the Y residue of the TPY motif. One of these peptide substrates was used in the development of a fluorescence polarization-based assay using an antibody as a sensor. The assay was successfully miniaturized for use with conventional fluorescence polarization (FP) reader technology in 8.5 microl and on the single microl scale using Evotec proprietary 2-dimensional fluorescence intensity distribution analysis (2D-FIDA) anisotropy and liquid handling technology. The steady-state kinetic parameters derived using the FP or 2D-FIDA anisotropy format assays correlated well with those generated using a radiometric assay. Moreover, the quantitative sensitivity to known inhibitors was maintained independent of the format and assay volume. In addition, the authors found that the 2D-FIDA anisotropy assay exhibited superior performance statistics (typical Z' = approximately 0.5) relative to conventional FP (typical Z' = 0.3) and yielded the additional benefit of order-of-magnitude savings in terms of reagent costs. The 2D-FIDA anisotropy assay was used to carry out a successful high-throughput screening in 1-microl final volume against company file compounds.     

Complete replacement of basic amino acid residues with cysteines in Rickettsia prowazekii ATP/ADP translocase

The ATP/ADP translocase (Tlc) of Rickettsia prowazekii is a basic protein with isoelectric point (pI)=9.84. It is conceivable, therefore, that basic residues in this protein are involved in electrostatic interactions with negatively charged substrates. We tested this hypothesis by individually mutating all basic residues in Tlc to Cys. Unexpectedly, mutations of only 20 out of 51 basic residues resulted in greater than 80% inhibition of transport activity. Moreover, 12 of 51Cys-substitution mutants exhibited higher than wild-type (WT) activity. At least in one case this up-effect was additive and the double mutant Lys422Cys Lys427Cys transported ATP five-fold better than WT protein. Since in these two single mutants and in the corresponding double mutant K(m)'s were similar to that of WT protein, we conclude that Tlc may have evolved a mechanism that limits the transporter's exchange rate and that at least these two basic residues play a key role in that mechanism. Based on the alignment of 16 Tlc homologs, the loss of activity in the mutants poorly correlates with charge conservation within the Tlc family. Also, despite the presence of three positively charged and one negatively charged intramembrane residues, we have failed to identify potential charge pairs (salt bridges) by either charge reversal or charge neutralization approaches.     

A new identity for MLK3 as an NIMA-related,cell cycle-regulated kinase that is localized near centrosomes and influences microtubule organization

Although conserved counterparts for most proteins involved in the G(2)/M transition of the cell cycle have been found in all eukaryotes, a notable exception is the essential but functionally enigmatic fungal kinase NIMA. While a number of vertebrate kinases have been identified with catalytic domain homology to NIMA, none of these resemble NIMA within its extensive noncatalytic region, a region critical for NIMA function in Aspergillus nidulans. We used a bioinformatics approach to search for proteins with homology to the noncatalytic region of NIMA and identified mixed lineage kinase 3 (MLK3). MLK3 has been proposed to serve as a component in MAP kinase cascades, particularly those resulting in the activation of the c-Jun N-terminal kinase (JNK). Here we describe the first in-depth study of endogenous MLK3 and report that, like NIMA, MLK3 phosphorylation and activity are enhanced during G(2)/M, whereas JNK remains inactive. Coincident with the G(2)/M transition, a period marked by dramatic reorganization of the cytoplasmic microtubule network, endogenous MLK3 transiently disperses away from the centrosome and centrosomal-proximal sites where it is localized during interphase. Furthermore, when overexpressed, MLK3, like NIMA, localizes to the centrosomal region, induces profound disruption of cytoplasmic microtubules and a nuclear distortion phenotype that differs from mitotic chromosome condensation. Cellular depletion of MLK3 protein using siRNA technology results in an increased sensitivity to the microtubule-stabilizing agent taxol. Our studies suggest a new role for MLK3, separable from its function in the JNK pathway, that may contribute to promoting microtubule instability, a hallmark of M phase entry.     

Utility of genetic screening in hypertrophic cardiomyopathy: prevalence and significance of novel and double (homozygous and heterozygous) beta-myosin mutations

Genetic screening of the beta-myosin heavy chain gene (MYH7) was evaluated in 100 consecutive unrelated patients with hypertrophic cardiomyopathy (HCM) and 200 normal unrelated subjects. Seventeen beta-myosin mutations were identified in 19 patients. Notably, 13, or 76%, were novel. Mutations were detected in both alleles in two patients: homozygous for Lys207Gln in one, and heterozygous for Pro211 Leu and Arg663His in another. No mutation was detected in the controls. MYH7-associated HCM was associated with more marked left atrial enlargement and syncope than non-MYH7-related HCM. Our findings indicate that: (1) screening methods should allow identification of novel mutations; and (2) more than one sarcomeric mutation may be present in a patient more commonly than is appreciated. Further studies are necessary to ascertain the clinical consequences of the novel and compound gene abnormalities, and to determine whether correlating functional domain to phenotype provides more useful information about the clinical significance of the molecular defects.