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Extinction-induced spatial dispersion in the radial arm maze: arrest by ethanol.

Adaptive changes in response to extinction can be observed if provisions are made that permit behavior to shift away from trained routine. In the present case, the baiting of four arms in the eight-arm radial maze increasingly restricted subject movements to those arms. The unbaited arms afforded a new direction for behavior to take during extinction. Withdrawal of reward was followed by an immediate and active expansion of sites visited. The previously unrewarded arms were now regularly sampled. This was one consequence of extinction. The other was the well-known decline in overall rate of arm entry, whether arms were previously baited or not. Ethanol, at doses of 1.5 or 2.0 g/kg, eliminated the spatial dispersion attendant upon nonreward. It did not affect the decline in overall responsiveness. Ethanol's deletion of extinction-induced spatial variability may account for its impairment on reversal and other kinds of tasks that require a shift away from old patterns of behavior.

Animals↗

Dynein arms are oscillating force generators.

Eukaryotic flagella beat rhythmically. Dynein is a protein that powers flagellar motion, and oscillation may be inherent to this protein. Here we determine whether oscillation is a property of dynein arms themselves or whether oscillation requires an intact axoneme, which is the central core of the flagellum and consists of a regular array of microtubules. Using optical trapping nanometry, we measured the force generated by a few dynein arms on an isolated doublet microtubule. When the dynein arms on the doublet microtubule contact a singlet microtubule and are activated by photolysis of caged ATP8, they generate a peak force of approximately 6pN and move the singlet microtubule over the doublet microtubule in a processive manner. The force and displacement oscillate with a peak-to-peak force and amplitude of approximately 2 pN and approximately 30 nm, respectively. The geometry of the interaction indicates that very few (possibly one) dynein arms are needed to generate the oscillation. The maximum frequency of the oscillation at 0.75 mM ATP is approximately 70 Hz; this frequency decreases as the ATP concentration decreases. A similar oscillatory force is also generated by inner dynein arms alone on doublet microtubules that are depleted of outer dynein arms. The oscillation of the dynein arm may be a basic mechanism underlying flagellar beating.

Adenosine Triphosphate↗

A protein extension to shorten RNA: elongated elongation factor-Tu recognizes the D-arm of T-armless tRNAs in nematode mitochondria.

Nematode mitochondria possess extremely truncated tRNAs. Of 22 tRNAs, 20 lack the entire T-arm. The T-arm is necessary for the binding of canonical tRNAs and EF (elongation factor)-Tu (thermo-unstable). The nematode mitochondrial translation system employs two different EF-Tu factors named EF-Tu1 and EF-Tu2. Our previous study showed that nematode Caenorhabditis elegans EF-Tu1 binds specifically to T-armless tRNA. C. elegans EF-Tu1 has a 57-amino acid C-terminal extension that is absent from canonical EF-Tu, and the T-arm-binding residues of canonical EF-Tu are not conserved. In this study, the recognition mechanism of T-armless tRNA by EF-Tu1 was investigated. Both modification interference assays and primer extension analysis of cross-linked ternary complexes revealed that EF-Tu1 interacts not only with the tRNA acceptor stem but also with the D-arm. This is the first example of an EF-Tu recognizing the D-arm of a tRNA. The binding activity of EF-Tu1 was impaired by deletion of only 14 residues from the C-terminus, indicating that the C-terminus of EF-Tu1 is required for its binding to T-armless tRNA. These results suggest that C. elegans EF-Tu1 recognizes the D-arm instead of the T-arm by a mechanism involving its C-terminal region. This study sheds light on the co-evolution of RNA and RNA-binding proteins in nematode mitochondria.

Amino Acid Sequence↗

Preoperative enzymo-histochemical diagnosis of dysganglionoses associated with anorectal malformations (ARM) with recto-vestibular and recto-perineal fistula.

The posterior sagittal anorectoplasty (PSARP) is widely recognized as the best technique available today for the surgical treatment of anorectal malformations (ARM). However, different retrospective studies on the functional results of PSARP in the treatment of ARM have shown different postoperative degrees of constipation. In particular, even in patients with normal sacrum, about 70% of operated ARM with vestibular fistula and about 50% with perineal fistula can be complicated by fecal constipation and pseudoincontinence. In order to identify preoperatively whether ARM patients present abnormal innervation patterns of rectal pouch and fistula (as reported by Holschneider et al [7]), we decided to study suction rectal biopsies performed by introducing SBT-100 rectal suction biopsy tool into the fistula at 6, 4, 3 and 2 cm from the meatus. To date, this approach has been adopted in 22 ARM cases (15 females and 7 males, age range 7 days-4 years), 6 of them with recto-vestibular fistula and 13 with recto-perineal fistula. Biopsies were frozen in isopentane at liquid nitrogen temperature and cryostatic sections were studied by acetylcholinesterase (AChE), succinic-dehydrogenase (SDH) and alpha-naphthyl-esterase (ANE) enzymo-histochemical techniques. The results concerning the innervation-type of fistula and proximal rectal pouch were confirmed by the biopsies obtained during PSARP. Our overall incidence of rectal innervation intrinsic disorders was 81.82%. In particular, all our cases of vestibular fistula presented associated dysganglionoses. The incidence of associated Hirschsprung's disease was high, corresponding to 18% of cases. Our results suggest that the high frequency of constipation in low forms of ARMS depends on primary intestinal neuronal malformations and it cannot be ascribed to a denervation secondary to rectal dissection and to PSARP procedure. We propose the introduction of this type of preoperative enzymo-histochemical diagnosis in ARM cases because it can select those patients with severe associated dysganglionoses. In our opinion, if this diagnosis is available preoperatively, PSARP can be performed without using abnormally innervated structures and reducing postoperative functional complications.

Anal Canal↗

Identification of dynein as the outer arms of sea urchin sperm axonemes.

The location of dynein, the main flagellar ATPase, within the sea urchin sperm axoneme was investigated by the use of immunofluorescence and immunoelectron microscopy, employing an antiserum against a tryptic fragment of dynein 1 (Fragment 1A) purified from sea urchin sperm flagella. The axonemes were found to be stained with the antiserum when examined by an indirect immunofluorescence technique. Immunoelectron microscopy with the antiserum and a ferritin-conjugated IgG fraction of goat antiserum to rabbit IgG revealed that, among the structures within the axoneme, only the outer arms were labeled with ferritin particles. With either the normal serum or antiserum absorbed with Fragment 1A, there were no ferritin particles within the axonemes. When the outer arms were extracted with 0.5 M NaCl, leaving the inner arms intact, again no ferritin dots were detected. Furthermore, it was found that the outer arm on the no. 5 doublet microtubule, which connects with the extra arm projection backward from the no. 6 doublet, had no attached ferritin particles. From these observations, it can be concluded that the outer arm consists of dynein (at least dynein 1) and that Fragment 1A, containing the active site for ATPase activity of dynein 1, is located at the distal end of the outer arms. The significance of the present findings is considered in connection with flagellar movement.

Adenosine Triphosphatases↗

Regional assignment of genes for human alpha-globin and phosphoglycollate phosphatase to the short arm of chromosome 16.

The human alpha-globin and phosphoglycollate phosphatase (EC 3.1.3.18) genes have been regionally localized to the short arm of human chromosome 16 (HC16). This was accomplished by fusing mouse fibroblasts (A9) to human fibroblasts that contain a reciprocal translocation between the long arms of chromosomes 16 and 11. The murine A9 cells are deficient in adenine phosphoribosyltransferase (APRT), an enzyme present on the long arm of HC16 (HC16q). Hybrid cells were grown in selection culture medium that required the cells to retain human APRT. Therefore, the hybrids exhibited stable retention of the entire HC16 or the rearranged chromosome containing HC16q. We isolated five independent primary and secondary hybrid cell lines which retained either HC16 or HC16q at a high frequency. The presence of human alpha-globin genes in the various clones was established directly by DNA extraction and hybridization to a cDNA probe for human alpha-globin genes. Autoradiographs showed that hybrid cells containing the long arm, but not the short arm, of HC16 showed only the background mouse bands. Hybrid cells that retained the entire HC16 demonstrated the band(s) containing the human alpha-globin genes. Hybrid cells that contained HC16 with its alpha-globin genes were then placed in culture medium that contained diaminopurine, which is lethal for cells containing APRT. These counter-selected hybrid cells had lost HC16 and also lost the human alpha-globin genes as determined by blot hybridization. The presence of alpha-globin gene sequences in the hybrid clones was concordant with HC16 only and not with any other human chromosome. These results confirm the assignment of alpha-globin genes to HC16 and localize the genes to the short arm. We also assign the locus for phosphoglycollate to the short arm of HC16.

Animals↗

Operator binding by lambda repressor heterodimers with one or two N-terminal arms.

The first 6 amino acids (NH2-Ser1-Thr2-Lys3-Lys4-Lys5-Pro6) of bacteriophage lambda cI repressor form a flexible arm that wraps around the operator DNA. Homodimeric lambda repressor has two arms. To determine whether both arms are necessary or only one arm is sufficient for operator binding, we constructed heterodimeric repressors with two, one, or no arms by fusing the DNA binding domain of lambda repressor to leucine zippers from Fos and Jun. Although only one arm is visible in the cocrystal structure of the N-domain-operator complex, our results indicate that both arms are required for optimal operator binding and normal site discrimination.

Amino Acid Sequence↗

Interactions between goal-directed eye and arm movements: arguments for an interdependent motor control.

The accuracy of movements of the arm directed toward a point in space was investigated in healthy human subjects. To study the influence of the eye movement itself, on the guidance of the arm in the absence of any visual context, subjects performed the goal-directed arm movements without visual feedback about the arm displacement and the target position. The subjects were asked either to keep their eyes centered or oriented toward a previously flashed target. The analysis of the distribution of the errors in arm final position in the two conditions suggests that the eye movement influences the final position adopted by the arm. It is postulated that an interaction exists between the eye and arm systems during the motor program elaboration phase.

Journal Article↗

Something sinistral going on? Asymmetry in arm movement frequency.

In a test of the hypothesis that the dominant arm would be more active than the nondominant arm, 70 young adults (including 34 left-handers) wore instrumented motion recorders on both wrists for 48 hours. Participants' left arms made approximately 80 more movements per hour than did their right arms, which was unexpected. This mean difference reflected a generalised leftward shift in the distribution of the frequency of arm movements. Two-thirds of the individuals in the sample were characterised by a sinistral bias in arm movement frequencies. Degree of sinistral bias in arm movement frequency was unrelated to hand preference as measured by the Waterloo Inventory. Implications of this new asymmetry are discussed.

Journal Article↗

Microtubule sliding in mutant Chlamydomonas axonemes devoid of outer or inner dynein arms.

To clarify the functional differentiation between the outer and inner dynein arms in eukaryotic flagella, their mechanochemical properties were assessed by measuring the sliding velocities of outer-doublet microtubules in disintegrating axonemes of Chlamydomonas, using wild-type and mutant strains that lack either of the arms. A special procedure was developed to induce sliding disintegration in Chlamydomonas axonemes which is difficult to achieve by ordinary methods. The flagella were first fragmented by sonication, demembranated by Nonidet P-40, and then perfused under a microscope with Mg-ATP and nagarse, a bacterial protease with broad substrate specificity. The sliding velocity varied with the Mg-ATP concentration in a Michaelis-Menten manner in the axonemes from the wild type and a motile mutant lacking the outer dynein arm (oda38). The maximal sliding velocity and apparent Michaelis constant for Mg-ATP were measured to be 13.2 +/- 1.0 micron/s and 158 +/- 36 microM for the wild type and 2.0 +/- 0.1 micron/s and 64 +/- 18 microM for oda38. These maximal sliding velocities were significantly smaller than those estimated in beating axonemes; the reason is not clear. The velocities in the presence or absence of 10(-5) M Ca2+ did not differ noticeably. The axonemes of nonmotile mutants lacking either outer arms (pf13A, pf22) or inner arms (pf23) were examined for their ability to undergo sliding disintegration in the presence of 0.1 mM Mg-ATP. Whereas pf13A axonemes underwent normal sliding disintegration, the other two species displayed it only very poorly. The poor ability of pf23 axonemes to undergo sliding disintegration raises the possibility that the outer dynein arm cannot function well in the absence of the inner arm.

Adenosine Triphosphatases↗

On the need to revise the arm structure in stylized anthropomorphic phantoms in lateral photon irradiation geometry.

Distributions of radiation absorbed dose within human anatomy have been estimated through Monte Carlo radiation transport techniques implemented for two different classes of computational anthropomorphic phantoms: (1) mathematical equation-based stylized phantoms and (2) tomographic image-based voxel phantoms. Voxel phantoms constructed from tomographic images of real human anatomy have been actively developed since the late 1980s to overcome the anatomical approximations necessary with stylized phantoms, which themselves have been utilized since the mid 1960s. However, revisions of stylized phantoms have also been pursued in parallel to the development of voxel phantoms since voxel phantoms (1) are initially restricted to the individual-specific anatomy of the person originally imaged, (2) must be restructured on an organ-by-organ basis to conform to reference individual anatomy and (3) cannot easily represent very fine anatomical structures and tissue layers that are thinner than the voxel dimensions of the overall phantom. Although efforts have been made to improve the anatomic realism of stylized phantoms, most of these efforts have been limited to attempts to alter internal organ structures. Aside from the internal organs, the exterior shapes, and especially the arm structures, of stylized phantoms are also far from realistic descriptions of human anatomy, and may cause dosimetry errors in the calculation of organ-absorbed doses for external irradiation scenarios. The present study was intended to highlight the need to revise the existing arm structure within stylized phantoms by comparing organ doses of stylized adult phantoms with those from three adult voxel phantoms in the lateral photon irradiation geometry. The representative stylized phantom, the adult phantom of the Oak Ridge National Laboratory (ORNL) series and two adult male voxel phantoms, KTMAN-2 and VOXTISS8, were employed for Monte Carlo dose calculation, and data from another voxel phantom, VIP-Man, were obtained from literature sources. The absorbed doses for lungs, oesophagus, liver and kidneys that could be affected by arm structures in the lateral irradiation geometry were obtained for both classes of phantoms in lateral monoenergetic photon irradiation geometries. As expected, those organs in the ORNL phantoms received apparently higher absorbed doses than those in the voxel phantoms. The overestimation is mainly attributed to the relatively poor representation of the arm structure in the ORNL phantom in which the arm bones are embedded within the regions describing the phantom's torso. The results of this study suggest that the overestimation of organ doses, due to unrealistic arm representation, should be taken into account when stylized phantoms are employed for equivalent or effective dose estimates, especially in the case of an irradiation scenario with dominating lateral exposure. For such a reason, the stylized phantom arm structure definition should be revised in order to obtain more realistic evaluations.

Algorithms↗

Combine-ARMS: a rapid and cost-effective protocol for molecular characterization of beta-thalassemia in Malaysia.

Beta-thalassemia major patients have chronic anemia and are dependent on blood transfusions to sustain life. Molecular characterization and prenatal diagnosis of beta3-thalassemia is essential in Malaysia because about 4.5% of the population are heterozygous carriers for beta-thalassemia. The high percentage of compound heterozygosity (47.62%) found in beta-thalassemia major patients in the Thalassaemia Registry, University of Malaya Medical Centre (UMMC), Malaysia, also supports a need for rapid, economical, and sensitive protocols for the detection of beta-thalassemia mutations. Molecular characterization of beta-thalassemia mutations in Malaysia is currently carried out using ARMS, which detects a single beta-thalassemia mutation per PCR reaction. We developed and evaluated Combine amplification refractory mutation system (C-ARMS) techniques for efficient molecular detection of two to three beta-thalassemia mutations in a single PCR reaction. Three C-ARMS protocols were evaluated and established for molecular characterization of common beta-thalassemia mutations in the Malay and Chinese ethnic groups in Malaysia. Two C-ARMS protocols (cd 41-42/IVSII #654 and -29/cd 71-72) detected the beta-thalassemia mutations in 74.98% of the Chinese patients studied. The CARMS for cd 41-42/IVSII #654 detected beta-thalassemia mutations in 72% of the Chinese families. C-ARMS for cd 41-42/IVSI #5/cd 17 allowed detection of beta-thalassemia mutations in 36.53% of beta-thalassemia in the Malay patients. C-ARMS for cd 41-42/IVSI #5/cd 17 detected beta-thalassemia in 45.54% of the Chinese patients. We conclude that C-ARMS with the ability to detect two to three mutations in a single reaction provides more rapid and cost-effective protocols for beta-thalassemia prenatal diagnosis and molecular analysis programs in Malaysia.

Asian People↗

A multiplex ARMS test for 10 cystic fibrosis (CF) mutations: evaluation in a prenatal CF screening program.

In Maine, prenatal screening for cystic fibrosis (CF) is offered through primary care providers. Cheekbrush (buccal) samples are routinely tested for eight mutations by multiplex PCR amplification of five exons, followed by dot-blot hybridization with pooled allele-specific oligonucleotides (ASO). The ASO methodology is widely used and effective, but somewhat time and labor intensive when applied to CF carrier testing or couple-based prenatal screening in the general pregnant population. Amplification Refractory Mutation System (ARMS) is an improvement of the PCR that allows rapid detection of mutations involving single base changes or small deletions/insertions. In this study, two multiplex ARMS reactions are used to test for 10 common CF mutations. Clinical evaluation of the ARMS test includes a retrospective study of 140 banked samples (54 cell line, proficiency testing, and buccal controls; 86 clinical buccal samples) with known CF genotype (57 with CF mutations, 83 no mutation), followed by a prospective trial in which 309 buccal samples are analyzed con-currently using both methods. The success rate of the ARMS test in buccal lysates is comparable to the ASO method; all CF mutations are successfully identified. For testing nonsterile buccal lysates with low DNA concentrations, optimized performance in the ARMS method is obtained using Amplitaq Gold polymerase. The ARMS method developed is easy, rapid (1 day), and avoids the need for ASO probe labeling, dot-blotting and autoradiography. This study provides further evidence that ARMS methodology is suitable for clinical CF mutation analysis.

Cheek↗

A ribozyme with DNA in the hybridising arms displays enhanced cleavage ability.

Hammerhead ribozymes cleave RNA substrates containing the UX sequence, where X = U, C or A, embedded within sequences which are complementary to the hybridising 'arms' of the ribozyme. In this study we have replaced the RNA in the hybridising arms of the ribozyme with DNA, and the resulting ribozyme is many times more active than its precursor. In turnover-kinetics experiments with a 13-mer RNA substrate, the kcat/Km ratios are 10 and 150 microM-1min-1 for the RNA- and DNA-armed ribozymes, respectively. The effect is due mainly to differences in kcat. In independent experiments where the cleavage step is rate-limiting, the DNA-armed ribozyme cleaves the substrate with a rate constant more than 3 times greater than the all-RNA ribozyme. DNA substrates containing a ribocytidine at the cleavage site have been shown to be cleaved less efficiently than their all-RNA analogues; again however, the DNA-armed ribozyme is more effective than the all-RNA ribozyme against such DNA substrates. These results demonstrate that there are no 2'-hydroxyl groups in the arms of the ribozyme that are required for cleavage; and that the structure of the complex formed by the DNA-armed ribozyme with its substrate is more favourable for cleavage than that formed by the all-RNA ribozyme and its substrate.

Animals↗

Analysis of complex genetic systems by ARMS-SSCP: application to HLA genotyping.

We have developed a new method for analysis of complex genetic systems by a combination of the Amplification Refractory Mutation System (ARMS) and Single-Strand Conformation Polymorphism (SSCP) analysis: ARMS-SSCP. Thus, a complex allelic series is subdivided into a number of groups by ARMS followed by the identification of specific alleles using SSCP analysis. We have shown that the HLA alleles at the DRB3 and DQB1 loci were distinguishable from each other using ARMS-SSCP. In 36 individuals typed for the DRB3 and 48 individuals typed for the DQB1 loci, ARMS-SSCP results were in complete agreement with those obtained using the established method of sequence-specific oligonucleotide (SSO) hybridisation. With silver staining, ARMS-SSCP is a rapid, non-radioactive and reliable method which also offers the possibility for detecting new HLA alleles. We have demonstrated that ARMS-SSCP can be performed using fluorescent PCR primers, a feature which gives the method potential for automation.

Base Sequence↗

Modification at position 9 with 1-methyladenosine is crucial for structure and function of nematode mitochondrial tRNAs lacking the entire T-arm.

The mitochondria of the nematode Ascaris suum have tRNAs with unusual secondary structures that lack either the T-arm or D-arm found in most other organisms. Of the twenty-two tRNA species present in the mitochondria of A.suum, twenty lack the entire T-arm and two serine tRNAs lack the D-arm. To understand how such unusual tRNAs work in the nematode mitochondrial translation system, we analyzed post-transcriptional modifications of 11 mitochondrial tRNA species purified from A.suum, 10 of which lacked a T-arm and one of which lacked a D-arm. The most characteristic feature of nematode mitochondrial tRNAs lacking a T-arm was the presence of 1-methyladenosine at position 9 (m1A9). Synthesis of T-armless tRNAs with or without the modified nucleoside showed that T-armless tRNAs without the modification had much lower aminoacylation and EF-Tu-binding activities than native tRNAs. The addition of a single methyl group to A9 of these tRNAs was sufficient to restore nearly native levels of aminoacylation and EF-Tu-binding activity as well as tertiary structure, suggesting that m1A9 is a key residue for the activity of T-armless tRNAs. Thus, m1A9 is indispensable for the structure and function of T-armless tRNAs of nematode mitochondrial origin.

Adenosine↗

An evaluation of the analgesic efficacy of intravenous regional anesthesia with lidocaine and ketorolac using a forearm versus upper arm tourniquet.

UNLABELLED: Intravenous regional anesthesia (IVRA) using a forearm tourniquet may be a potentially safer technique compared with using an upper arm tourniquet. Ketorolac is a useful adjuvant to lidocaine for IVRA. In this study, we assessed the analgesic efficacy of administering IVRA lidocaine and ketorolac with either a forearm or upper arm tourniquet for outpatient hand surgery. Upper arm IVRA was established using 40 mL of a solution containing 200 mg of lidocaine and ketorolac 20 mg (0.5 mg/mL). Forearm IVRA was established using 20 mL of a solution containing 100 mg of lidocaine and ketorolac 10 mg (0.5 mg/mL). Onset and duration of sensory block as well as postoperative pain and analgesic use were recorded. The patients who received forearm IVRA had a significantly longer period during which they required no analgesics (701 +/- 133 min) compared with 624 +/- 80 min for the upper arm IVRA ketorolac patients (P = 0.032). Onset of sensory block was similar between the two groups; however, recovery of sensation was significantly longer in the Forearm IVRA (22 +/- 5 min) group compared with the Upper Arm IVRA (13 +/- 3 min) group (P < 0.05). There were no differences in postoperative analgesic use or pain scores between the two groups. We conclude that forearm IVRA with lidocaine and ketorolac provides safe and effective perioperative analgesia for patients undergoing ambulatory hand surgery. This technique results in a longer duration of sensory block and prolonged postoperative analgesia compared with upper arm IVRA while using one-half the doses of both lidocaine and ketorolac. IMPLICATIONS: Forearm tourniquet intravenous regional anesthesia (IVRA) with 50% less lidocaine and ketorolac provides for both a longer duration of sensory block and prolonged postoperative analgesia compared with upper arm IVRA.

Adult↗

Moment arm lengths of trunk muscles to the lumbosacral joint obtained in vivo with computed tomography.

Computed tomography is valuable in obtaining in vivo length of muscular moment arms, which can be used in biomechanical models. Using computed tomography, the authors measured lengths of the muscular moment arms of the erector spinae, psoas, oblique abdominal, and rectus abdominis muscles to the bilateral motion axis (about which flexion/extension movements occur) and to the anteroposterior motion axis (about which lateral bending occurs), both axes running through the lumbosacral joint. The subjects were 21 patients. Examinations were performed on a General Electric CT/T 7800 scanner. The average erector spinae moment arm to the bilateral motion axis was 68 mm, which is greater than the moment arm used earlier. The results also revealed significant differences in moment arm length between men and women for several muscles. The authors conclude that for some muscles different moment arm lengths should be used for men and for women. The moment arms reported are proposed for use in biomechanical models for calculating lumbosacral joint load.

Aged↗