Interaction between attention demanding motor and cognitive tasks and static postural stability.

BACKGROUND: Due to the often-reported decrease in postural stability in the elderly, it is important to understand factors that may contribute to reduced postural stability. It is possible that attention-demanding focal tasks performed concurrent with postural regulation influence postural stability. OBJECTIVE: This study utilized dual-task methodology to determine if motor or cognitive focal tasks interact with center of pressure (COP) excursion during static bipedal stance in healthy young and healthy elderly subjects (n = 18). METHODS: The cognitive task involved silently solving an orally-presented multi-step arithmetic problem over a 30-second period. The motor task was a 30-second bilateral static finger-thumb pinch task performed at 10% of maximal voluntary contraction with a pair of pinch-force transducers. Each focal task was performed separately, and in a condition in which both tasks were performed simultaneously. COP excursion was compared in quiet standing (no focal task) and during performance of the focal tasks with full vision and with vision occluded. RESULTS: Performance on the focal tasks was unaffected by increased postural demands during stance as compared to a seated baseline condition. This was the case for both age groups, and for the full vision and occluded vision conditions. Medio-lateral COP excursion was reduced over the quiet standing pretest condition when attentional focus was on the cognitive task, suggesting that COP was influenced centrally during cognition. In contrast, COP excursion increased over the quiet standing pretest condition when performing the motor focal task, suggesting a reduced ability to suppress sway when the motor system was concurrently occupied with a voluntary task that shared the same input-output resources. CONCLUSION: The ability to share attentional resources among focal and postural tasks was similar in healthy young and elderly subjects.

Specific identification of three low molecular weight membrane-associated antigens of Helicobacter pylori.

BACKGROUND: A large number of Helicobacter pylori proteins are antigenic, but antibodies to these proteins persist in spite of the eradication of the infection. METHODS AND RESULTS: The analysis of sera from H. pylori-infected and non-infected patients, before and 3 and 5 months after eradication, showed that the antibody response against unknown H. pylori antigens at 32, 30, 22 and 14 kDa in sodium dodecylsulphate polyacrylamide gel electrophoresis decreased by > or = 60% at 3 months and > or = 70% at 5 months after treatment. Two-dimensional gel electrophoresis and mass spectrometry allowed the identification of eight proteins at these positions: neuraminyl-lactose-binding haemagglutinin precursor, 3-oxoadipate CoA-transferase subunit A, elongation factor P, peptidoglycan-associated lipoprotein precursor, hypothetical protein HP0596, adhesin-thiol peroxidase, 50S ribosomal protein L7/L12 and subunit b' of the F(0) ATP synthase. Three of these eight, expressed as recombinant proteins (32 kDa neuraminyl-lactose-binding haemagglutinin precursor, 30 kDa peptidoglycan-associated lipoprotein precursor and 22 kDa hypothetical protein HP0596), reacted specifically with sera from infected patients, while the 14 kDa 50S ribosomal protein L7/L12 cross-reacted with one out of five sera from H. pylori-negative patients. The other recombinant proteins did not show significant immunoreactivity. CONCLUSIONS: Four low molecular weight antigens were identified by these methods, three of which were specific. Immunoreaction with these three proteins (neuraminyl-lactose-binding haemagglutinin precursor, peptidoglycan-associated lipoprotein precursor and hypothetical protein HP0596) could provide a serological assessment not only of H. pylori infection, but also of eradication.

Chromosome targeting at short polypurine sites by cationic triplex-forming oligonucleotides.

Triplex-forming oligonucleotides (TFOs) bind specifically to duplex DNA and provide a strategy for site-directed modification of genomic DNA. Recently we demonstrated TFO-mediated targeted gene knockout following systemic administration in animals. However, a limitation to this approach is the requirement for a polypurine tract (typically 15-30 base pairs (bp)) in the target DNA to afford high affinity third strand binding, thus restricting the number of sites available for effective targeting. To overcome this limitation, we have investigated the ability of chemically modified TFOs to target a short (10 bp) site in a chromosomal locus in mouse cells and induce site-specific mutations. We report that replacement of the phosphodiester backbone with cationic phosphoramidate linkages, either N,N-diethylethylenediamine or N,N-dimethylaminopropylamine, in a 10-nucleotide, psoralen-conjugated TFO confers substantial increases in binding affinity in vitro and is required to achieve targeted modification of a chromosomal reporter gene in mammalian cells. The triplex-directed, site-specific induction of mutagenesis in the chromosomal target was charge- and modification-dependent, with the activity of N,N-diethylethylenediamine > N,N-dimethylaminopropylamine phosphodiester, resulting in 10-, 6-, and <2-fold induction of target gene mutagenesis, respectively. Similarly, N,N-diethylethylenediamine and N,N-dimethylaminopropylamine TFOs were found to enhance targeting at a 16-bp G:C bp-rich target site in a chromatinized episomal target in monkey COS cells, although this longer site was also targetable by a phosphodiester TFO. These results indicate that replacement of phosphodiester bonds with positively charged N,N-diethylethylenediamine linkages enhances intracellular activity and allows targeting of relatively short polypurine sites, thereby substantially expanding the number of potential triplex target sites in the genome.

Sites of pH regulation of the urea channel of Helicobacter pylori.

Helicobacter pylori (Hp) and Streptococcus salivarius (Ss) require intrabacterial urease for acid resistance and express a urea channel, UreI. The presence of UreI was shown to increase urea permeability approximately 300-fold over that of a non-polar ureI deletion mutant. Expression of SsUreI in Xenopus oocytes increased urea uptake pH independently, whereas HpUreI shows an acidic pH dependence, half-maximal at pH 6.0. Mutagenesis of all histidines, aspartates, glutamates and the lysine in the periplasmic domain of HpUreI showed that His-123, His-131, Asp-129, Asp-140, Glu-138 and Lys-132 in the second periplasmic loop (PL2) and His-193 in the C-terminus (Ct) were important for activation of transport. With the exception of a lysine that was shown to substitute for His-193 in HpUreI, these charged amino acids are absent in SsUreI. A chimera in which PL1 of HpUreI was replaced by PL1 of SsUreI retained activity at acidic pH and gained partial activity at neutral pH. Exchange of PL2 inactivated transport, whereas exchange of Ct had no effect. Chimeras, in which either PL1 or PL2 of HpUreI replaced those of SsUreI, retained wild-type transport, but replacement of the Ct or both loops inactivated transport. PL1 appears to be important for restricting transport through HpUreI at neutral pH, whereas protonation of three histidines in PL2 and Ct and the presence of three dicarboxylic amino acids in PL2 appears to be necessary to activate HpUreI at acidic pH.

Oligonucleotide-based strategies to reduce gene expression.

Research on embryonic development and differentiation provides a sensitive, but challenging opportunity to use a variety of techniques designed to modulate gene expression. Changes in the expression of a single gene can alter levels of other genes and provide information on developmentally regulated gene expression pathways. The morphological consequences of altered gene expression can link gene expression to developmental fate. Oligonucleotide-based approaches offer a variety of means to potentially disrupt normal gene expression. The basis for some of these approaches is presented in this review.

Distinct roles for TBP and TBP-like factor in early embryonic gene transcription in Xenopus.

The TATA-binding protein (TBP) is believed to function as a key component of the general transcription machinery. We tested the role of TBP during the onset of embryonic transcription by antisense oligonucleotide-mediated turnover of maternal TBP messenger RNA. Embryos without detectable TBP initiated gastrulation but died before completing gastrulation. The expression of many genes transcribed by RNA polymerase II and III was reduced; however, some genes were transcribed with an efficiency identical to that of TBP-containing embryos. Using a similar antisense strategy, we found that the TBP-like factor TLF/TRF2 is essential for development past the mid-blastula stage. Because TBP and a TLF factor play complementary roles in embryonic development, our results indicate that although similar mechanistic roles exist in common, TBP and TLF function differentially to control transcription of specific genes.

Review article: the control of gastric acid and Helicobacter pylori eradication.

This review focuses on the gastric acid pump as a therapeutic target for the control of acid secretion in peptic ulcer and gastro-oesophageal reflux disease. The mechanism of the proton pump inhibitors is discussed as well as their clinical use. The biology of Helicobacter pylori as a gastric denizen is then discussed, with special regard to its mechanisms of acid resistance. Here the properties of the products of the urease gene clusters, ureA, B and ureI, E, F, G and H are explored in order to explain the unique location of this pathogen. The dominant requirement for acid resistance is the presence of a proton gated urea transporter, UreI, which increases access of gastric juice urea to the intrabacterial urease 300-fold. This enables rapid and continuous buffering of the bacterial periplasm to approximately pH 6.0, allowing acid resistance and growth at acidic pH in the presence of 1 mM urea. A hypothesis for the basis of combination therapy for eradication is also presented.

Contrast-enhanced immunoelectron microscopy for Helicobacter pylori.

Since a method of contrast enhancement for immunoelectron microscopy has not been available in bacteriology, the morphological localization of proteins of Helicobacter pylori is not well known. In this report, we established a method of contrast enhancement in immunoelectron microscopy in this organism. Immunostained ultrathin sections are stained with a mixture of alcian blue and osmium tetroxide prior to staining with uranyl acetate. This method of staining provided good contrast enhancement of the bacterial cell wall and membrane without any loss of immunolabeled gold particles on the ultrathin section.

Precision-grip force changes in the anatomical and prosthetic limb during predictable load increases.

This study examined precision-grip force applied to an instrumented test object held aloft while the weight of the object was predictably varied by transporting and placing loads (50, 100, or 200 g) atop the test object. Transport of the loads was performed either by the subject or the experimenter. Grip force was examined in four non-amputee control subjects and in the anatomical and prosthetic hand of a subject with a prosthetic device. As subjects transported the load, anticipatory grip-force changes occurred in the anatomical hands and prosthetic hand, which were scaled in relation to the load. When the experimenter transported the load to the anatomical hands of control subjects or the prosthetic user, anticipatory increases in grip force occurred that also were scaled in relation to load. However, when the experimenter transported the load to the prosthetic hand, anticipatory grip-force adjustments were absent. During the phase in which the load was being assumed by the postural hand, grip forces in the anatomical hands and prosthetic hand were further scaled to load demands. Ability to adapt grip force in the prosthetic hand during this phase suggested that the subject was utilizing sensory information from the residual limb to adjust grip force. Thus, while anticipatory changes precede the process of adaptation to load changes, actual sensory consequences resulting from added weight remain necessary to fully adapt grip force to load demands, even for the prosthetic user.

The interaction of observational learning with overt practice: effects on motor skill learning.

This study explored various methods of combining observational learning via demonstration with the effects of overt practice for learning a discrete action pattern. Three groups were compared that varied by the timing of demonstration in relation to practice. An all-pre-practice demonstration group viewed 10 pre-practice videotape demonstrations of an expert performing the skill, and then engaged in practice. An interspersed demonstration group viewed one pre-practice demonstration, then initiated practice on the skill. Every three attempts, practice was halted while participants viewed another demonstration, with this pattern repeated throughout acquisition. A combination demonstration group experienced elements of each schedule by viewing five demonstrations prior to practice, then five more once practice had begun (one every three attempts) so that modeling was completed by mid-acquisition. Ratings of form and accuracy were assessed in an acquisition phase, an immediate retention test, and a 48-h retention test. Group main effects for form scores were detected in acquisition, immediate, and 48-h retention, with the combination group obtaining the highest form scores, followed by the all-pre-practice group, and finally the interspersed group. These findings suggest that several modeling exposures before practice and several more exposures in the early stages of practice were optimal for acquisition and retention of form.

Targeted elimination of zygotic messages in Xenopus laevis embryos by modified oligonucleotides possessing terminal cationic linkages.

We have designed a new class of modified antisense oligodeoxyribonucleotides (ODN) consisting of a central contiguous stretch of 6-8 unmodified nucleotides flanked by 3'- and 5'-regions containing several nucleotides joined by cationic internucleoside linkages. The positive charge results from modification of the internucleoside linkages as N, N -diethylethylene-diamine phosphoramidates. These zwitterionic compounds show improved antisense activity in both Xenopus oocytes and embryos compared to our previously described chimeric oligonucleotides possessing neutral terminal internucleoside linkages. Using the localized maternal mRNA An2 as a target, we have shown that chimeric oligonucleotides with terminal positive charges are very effective in the sequence-specific elimination of maternal messages present in both oocytes and embryos. In addition, using the embryonic mRNA GS17 as a target, we have shown that these oligonucleotides can direct RNase H-mediated cleavage of messages produced at the onset of zygotic transcription, after the mid-blastula stage. These new compounds should be useful in attenuating embryonic gene expression to study the role of specific proteins in early vertebrate development.

Understanding oligonucleotide-mediated inhibition of gene expression in Xenopus laevis oocytes.

Triplex-forming oligonucleotides (TFOs) modified with N,N-diethylethylenediamine can inhibit the expression of a reporter plasmid in Xenopus oocytes if the triplex is preformed prior to injection while unmodified oligonucleotides cannot. Here we show that merely forming a triplex in a reporter plasmid does not disrupt transcription, but when TFOs are targeted to sites within the transcribed region of a reporter gene then gene activity is inhibited. TFO-based inhibition did not lead to large scale degradation or mutation of the reporter plasmid, but dramatically lowered mRNA levels. Finally, we investigated the accessibility of a triplex target site on a reporter plasmid after injection into nuclei. We found that the site used for our previous studies was inaccessible to restriction endonuclease after injection into nuclei. This observation may explain why inhibition was dependent on forming the triplex before injection into oocytes. Based on the assumption that oligonucleotide association, like restriction enzyme access, was excluded by nucleosome formation, additional target sites were inserted so that all sites could not simultaneously be associated with the octamer core of a nucleosome. With multiple target sites prior association of the plasmid with nuclear proteins does not prevent oligonucleotide-mediated inhibition of gene activity.

Confocal imaging of early heart development in Xenopus laevis.

Xenopus laevis provides a number of advantages to studies on cardiovascular development. The embryos are fairly large, are easy to obtain, and can develop at ambient temperature in simple buffer solutions. Although classic descriptions of heart development exist, the ability to use whole-mount immunohistochemical methods and confocal microscopy may enhance the ability to understand both normal and experimentally perturbed cardiovascular development. We have started to examine the early stages of cardiac development in Xenopus, seeking to identify antibodies and fixatives that allow easy examination of the developing heart. We have used monoclonal antibodies (mAbs) raised against bovine cardiac troponin T and chicken tropomyosin to visualize cardiac muscle, a goat antibody recognizing bovine type VI collagen to stain the lining of vessels, and the JB3 mAb raised against chicken fibrillin, which allows the visualization of a variety of cardiovascular tissues during early development. Results from embryonic stages 24-46 are presented.

A H+-gated urea channel: the link between Helicobacter pylori urease and gastric colonization.

Acidic media trigger cytoplasmic urease activity of the unique human gastric pathogen Helicobacter pylori. Deletion of ureI prevents this activation of cytoplasmic urease that is essential for bacterial acid resistance. UreI is an inner membrane protein with six transmembrane segments as shown by in vitro transcription/translation and membrane separation. Expression of UreI in Xenopus oocytes results in acid-stimulated urea uptake, with a pH profile similar to activation of cytoplasmic urease. Mutation of periplasmic histidine 123 abolishes stimulation. UreI-mediated transport is urea specific, passive, nonsaturable, nonelectrogenic, and temperature independent. UreI functions as a H+-gated urea channel regulating cytoplasmic urease that is essential for gastric survival and colonization.

Expression of the Helicobacter pylori ureI gene is required for acidic pH activation of cytoplasmic urease.

ureI encodes an integral cytoplasmic membrane protein. It is present in the urease gene cluster of Helicobacter pylori and is essential for infection and acid survival, but its role is unknown. To determine the function of UreI protein, we produced H. pylori ureI deletion mutants and measured the pH dependence of urease activity of intact and lysed bacteria and the effect of urea on the membrane potential. We also determined ureI expression, urease activity, and the effect of urea on membrane potential of several gastric and nongastric Helicobacter species. ureI was found to be present in the genome of the gastric Helicobacter species and absent in the nongastric Helicobacter species studied, as determined by PCR. Likewise, Western blot analysis confirmed that UreI was expressed only in the gastric Helicobacter species. When UreI is present, acidic medium pH activation of cytoplasmic urease is found, and urea addition increases membrane potential at acidic pH. The addition of a low concentration of detergent raised urease activity of intact bacteria at neutral pH to that of their homogenates, showing that urease activity was membrane limited. No acidic pH activation or urea induced membrane potential changes were found in the nongastric Helicobacter species. The ureI gene product is probably a pH activated urea transporter or perhaps regulates such a transporter as a function of periplasmic pH.

RanGTP-regulated interactions of CRM1 with nucleoporins and a shuttling DEAD-box helicase.

CRM1 is an export receptor mediating rapid nuclear exit of proteins and RNAs to the cytoplasm. CRM1 export cargoes include proteins with a leucine-rich nuclear export signal (NES) that bind directly to CRM1 in a trimeric complex with RanGTP. Using a quantitative CRM1-NES cargo binding assay, significant differences in affinity for CRM1 among natural NESs are demonstrated, suggesting that the steady-state nucleocytoplasmic distribution of shuttling proteins could be determined by the relative strengths of their NESs. We also show that a trimeric CRM1-NES-RanGTP complex is disassembled by RanBP1 in the presence of RanGAP, even though RanBP1 itself contains a leucine-rich NES. Selection of CRM1-binding proteins from Xenopus egg extract leads to the identification of an NES-containing DEAD-box helicase, An3, that continuously shuttles between the nucleus and the cytoplasm. In addition, we identify the Xenopus homologue of the nucleoporin CAN/Nup214 as a RanGTP- and NES cargo-specific binding site for CRM1, suggesting that this nucleoporin plays a role in export complex disassembly and/or CRM1 recycling.

Utilization of antisense oligodeoxynucleotides with embryonic tissues in culture.

Experimental embryology has long used manipulation of interacting tissues to examine questions of tissue interaction and differentiation. The potential for specific manipulation of gene expression in such tissues has made the utilization of antisense techniques desirable. However, problems with this methodology have discouraged many investigators from using this approach. Selection of target sequences for antisense oligonucleotides, delivery of oligonucleotides into cells or tissues, and the type of modification of the oligonucleotide to be used all present concerns that must be addressed. This paper describes our approach to selection of target sequence and methods of delivery and describes the synthesis of a methoxyethylamidate-modified antisense oligonucleotide that has proved useful in our studies. This approach has enabled us to explore aspects of tissue interaction in the embryonic heart that would have been difficult to explore in a genetic model.

TGFbeta2 and TGFbeta3 have separate and sequential activities during epithelial-mesenchymal cell transformation in the embryonic heart.

Heart valve formation is initiated by an epithelial-mesenchymal cell transformation (EMT) of endothelial cells in the atrioventricular (AV) canal. Mesenchymal cells formed from cardiac EMTs are the initial cellular components of the cardiac cushions and progenitors of valvular and septal fibroblasts. It has been shown that transforming growth factor beta (TGFbeta) mediates EMT in the AV canal, and TGFbeta1 and 2 isoforms are expressed in the mouse heart while TGFbeta 2 and 3 are expressed in the avian heart. Depletion of TGFbeta3 in avian or TGFbeta2 in mouse leads to developmental defects of heart tissue. These observations raise questions as to whether multiple TGFbeta isoforms participate in valve formation. In this study, we examined the localization and function of TGFbeta2 and TGFbeta3 in the chick heart during EMT. TGFbeta2 was present in both endothelium and myocardium before and after EMT. TGFbeta2 antibody inhibited endothelial cell-cell separation. In contrast, TGFbeta3 was present only in the myocardium before EMT and was in the endothelium at the initiation of EMT. TGFbeta3 antibodies inhibited mesenchymal cell formation and migration into the underlying matrix. Both TGFbeta2 and 3 increased fibrillin 2 expression. However, only TGFbeta2 treatment increased cell surface beta-1,4-galactosyltransferase expression. These data suggest that TGFbeta2 and TGFbeta3 are sequentially and separately involved in the process of EMT. TGFbeta2 mediates initial endothelial cell-cell separation while TGFbeta3 is required for the cell morphological change that enables the migration of cells into the underlying ECM.

Production of the transforming growth factor-beta binding protein endoglin is regulated during chick heart development.

The early embryonic heart consists of two cell types. The cells form an inner epithelial tube of endocardium within an outer tube of myocardium separated by a cell-free extracellular matrix. A crucial process in heart development is the production of cushion mesenchyme in the atrioventricular (AV) canal and outflow tract (OT). Cushion mesenchyme differentiates from the endocardium in response to signaling molecules produced by the adjacent myocardium. In chicken hearts, both transforming growth factor-beta3 (TGF-beta3) and TGF-beta2 are present and have been identified as being important in the production of cushion mesenchyme. We were interested in how the signals from these two similar molecules may be differentiated during early heart development. To this end, we examined the expression of endoglin, a TGF-beta receptor molecule, in the developing chick heart. Endoglin is typically located on endothelial cell layers and binds tightly to TGF-beta1 and TGF-beta3 but not well to TGF-beta2. We show that during the formation of the primitive heart tube, endoglin is found at relatively high levels in both presumptive myocardium and endocardium. However, as myocardium differentiates and development proceeds, endoglin expression is progressively reduced. At stage 20 in the heart, endoglin expression is most readily seen in the AV canal and the OT. This pattern of expression is similar to the reported TGF-beta3 expression patterns in the heart.