Length heterogeneity of the large spacer of Vicia faba rDNA is due to the differing number of a 325 bp repetitive sequence elements

Summary The DNA fragments including the whole large spacer region of Vicia faba rDNA were cloned in plasmid pBR325. Sixteen clones were classed into five groups which differed from each other in the lengths of the rDNA inserts. Physical maps of these length variants cloned were constructed using EcoRI, SalI, HpaI, MluI and AccI and evidence was obtained that the length heterogeneity was due mainly to the differing number of 325 base pairs (bp) subrepeating elements in the large spacer. Sequence analysis of this subrepeating element revealed that it consisted of a duplet of an approximately 155 bp sequence and a 14 bp unrelated sequence. This structure of the repetitive element is novel.     

Molecular Cloning and Restriction Analysis of EcoRI-fragments of Vicia faba rDNA

EcoRI-fragments of Vicia faba rDNA were cloned in plasmid pBR325.Southern blot hybridization of BamHI-digests of these clonedplasmids and Vicia genomic DNA led to the determination of relativepositions of BamHl sites in the rDNA and the physical map thathad been tentatively made is corrected. (Received May 20, 1982; Accepted July 13, 1983)     

Comparison of human and mouse E-selectin binding to Sialyl-Lewis<Superscript>x</Superscript>

Background

During inflammation, leukocytes are captured by the selectin family of adhesion receptors lining blood vessels to facilitate exit from the bloodstream. E-selectin is upregulated on stimulated endothelial cells and binds to several ligands on the surface of leukocytes. Selectin:ligand interactions are mediated in part by the interaction between the lectin domain and Sialyl-Lewis x (sLe^x), a tetrasaccharide common to selectin ligands. There is a high degree of homology between selectins of various species: about 72 and 60 % in the lectin and EGF domains, respectively. In this study, molecular dynamics, docking, and steered molecular dynamics simulations were used to compare the binding and dissociation mechanisms of sLe^x with mouse and human E-selectin. First, a mouse E-selectin homology model was generated using the human E-selectin crystal structure as a template.

Results

Mouse E-selectin was found to have a greater interdomain angle, which has been previously shown to correlate with stronger binding among selectins. sLe^x was docked onto human and mouse E-selectin, and the mouse complex was found to have a higher free energy of binding and a lower dissociation constant, suggesting stronger binding. The mouse complex had higher flexibility in a few key residues. Finally, steered molecular dynamics was used to dissociate the complexes at force loading rates of 2000–5000 pm/ps². The mouse complex took longer to dissociate at every force loading rate and the difference was statistically significant at 3000 pm/ps². When sLe^x-coated microspheres were perfused through microtubes coated with human or mouse E-selectin, the particles rolled more slowly on mouse E-selectin.

Conclusions

Both molecular dynamics simulations and microsphere adhesion experiments show that mouse E-selectin protein binds more strongly to sialyl Lewis x ligand than human E-selectin. This difference was explained by a greater interdomain angle for mouse E-selectin, and greater flexibility in key residues. Future work could introduce similar amino acid substitutions into the human E-selectin sequence to further modulate adhesion behavior.

     

Analysis of zebrafish Mhc using BAC clones

 Although major histocompatibility complex (Mhc) genes have been identified in a number of species, little is yet known about their organization in species other than human and mouse. The zebrafish, Danio rerio, is a good candidate for full elucidation of the organization of its Mhc. As a step toward achieving this goal, a commercially available zebrafish BAC library was screened with probes specific for previously identified zebrafish class I and class II genes, as well as for genes controlling the proteasome subunits LMP7 and LMP2. Restriction maps of the individual positive clones were prepared and the Mhc (LMP7) genes localized to specific fragments. The total length of genomic DNA fragments with Mhc genes was approximately 1700 kilobases (kb) (200 kb of fragments bearing class I loci and 1500 kb of fragments bearing class II loci). One of the two class I loci (Dare-UCA) is closely associated with the LMP7 locus; the second class I locus (Dare-UAA) is more than 50 kb distant from the UCA locus and has no LMP genes associated with it. None of the class II genes are linked to the class I or the LMP genes. All six of the previously identified class II B genes and one of the three class II A genes were found to be present in the BAC clones; no new Mhc loci could be identified in the library. Each of the six previously identified class II B loci was found to be borne by a separate group of BAC clones. The Dare-DAB and -DAA loci were found on the same clone, approximately 15 kb apart from each other. An expansion of DCB and DDB loci was detected: the zebrafish genome may contain at least five closely related DCB and two closely related DDB loci which are presumably the products of relatively recent tandem duplication. These results are consistent with linkage studies and indicate that in the zebrafish, the class I and class II loci are on different chromosomes, and the class II loci are in three different regions, at least two of which are on different chromosomes. Received: 14 August 1997 / Revised: 16 September 1997     

Age-related changes of the stretch reflex excitability in human ankle muscles

The purpose of this study was to characterize the effects of aging on the stretch reflex in the ankle muscles, and in particular to compare the effects on the ankle dorsi-flexor (tibialis anterior: TA) and the plantar-flexor (soleus: SOL). Stretch reflex responses were elicited in the TA and SOL at rest and during weak voluntary contractions in 20 elderly and 23 young volunteers. The results indicated that, in the TA muscle, the elderly group had a remarkably larger long-latency reflex (LLR), whereas no aging effect was found in the short latency reflex (SLR). These results were very different from those in the SOL muscle, which showed significant aging effects in the SLR and medium latency reflex (MLR), but not in the LLR. Given the fact that the LLR of the TA stretch reflex includes the cortical pathway, it is probable that the effects of aging on the TA stretch reflex involve alterations not only at the spinal level but also at the cortical level. The present results indicate that the stretch reflexes of each of the ankle antagonistic muscles are affected differently by aging, which might have relevance to the neural properties of each muscle.     

Traveling EEG slow oscillation along the dorsal attention network initiates spontaneous perceptual switching

An ambiguous figure such as the Necker cube causes spontaneous perceptual switching (SPS). The mechanism of SPS in multistable perception has not yet been determined. Although early psychological studies suggested that SPS may be caused by fatigue or satiation of orientation, the neural mechanism of SPS is still unknown. Functional magnetic resonance imaging (fMRI) has shown that the dorsal attention network (DAN), which mainly controls voluntary attention, is involved in bistable perception of the Necker cube. To determine whether neural dynamics along the DAN cause SPS, we performed simultaneous electroencephalography (EEG) and fMRI during an SPS task with the Necker cube, with every SPS reported by pressing a button. This EEG–fMRI integrated analysis showed that (a) 3–4 Hz spectral EEG power modulation at fronto-central, parietal, and centro-parietal electrode sites sequentially appeared from 750 to 350 ms prior to the button press; and (b) activations correlating with the EEG modulation traveled along the DAN from the frontal to the parietal regions. These findings suggest that slow oscillation initiates SPS through global dynamics along the attentional system such as the DAN.     

On the reflex coactivation of ankle flexor and extensor muscles induced by a sudden drop of support surface during walking in humans.

Recent studies have revealed that the stretch reflex responses of both ankle flexor and extensor muscles are coaugmented in the early stance phase of human walking, suggesting that these coaugmented reflex responses contribute to secure foot stabilization around the heel strike. To test whether the reflex responses mediated by the stretch reflex pathway are actually induced in both the ankle flexor and extensor muscles when the supportive surface is suddenly destabilized, we investigated the electromyographic (EMG) responses induced after a sudden drop of the supportive surface at the early stance phase of human walking. While subjects walked on a walkway, the specially designed movable supportive surface was unexpectedly dropped 10 mm during the early stance phase. The results showed that short-latency reflex EMG responses after the impact of the drop (<50 ms) were consistently observed in both the ankle flexor and extensor muscles in the perturbed leg. Of particular interest was that a distinct response appeared in the tibialis anterior muscle, although this muscle showed little background EMG activity during the stance phase. These results indicated that the reflex activities in the ankle muscles certainly acted when the supportive surface was unexpectedly destabilized just after the heel strike during walking. These reflex responses were most probably mediated by the facilitated stretch reflex pathways of the ankle muscles at the early stance phase and were suggested to be relevant to secure stabilization around the ankle joint during human walking.     

Intervention of somatic mutational events in vivo by a germline defect at the adenine phosphoribosyltransferase locus

Both germline and somatic mutations are known to affect phenotypes of human cells in vivo. In previous studies, we cloned mutant peripheral blood T cells from germline heterozygous humans for adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7) deficiency and found that approximately 1.3 × 10^–4 peripheral T cells had undergone in vivo somatic mutations. Loss of heterozygosity (LOH) was the major cause of the mutations at the APRT locus since approximately 80% of the mutant T cell clones exhibited loss of normal alleles. In the present study, we identified three heterozygous individuals for APRT deficiency (representing two separate families), in whom none of the somatic mutant cells exhibited LOH at the APRT locus. The germline mutant APRT alleles of these heterozygotes from two unrelated families had the same gross DNA abnormalities detectable by Southern blotting. None of the germline mutant APRT alleles so far reported had such gross DNA abnormalities. The data suggest that the germline mutation unique to these heterozygous individuals is associated with the abrogation of LOH in somatic cells. The absence of LOH at a different locus has already been reported in vitro in an established cell line but the present study describes the first such event in vivo in human individuals. Received: 10 May 1996     

Uncertainty of knee joint muscle activity during knee joint torque exertion: the significance of controlling adjacent joint torque.

In the single-joint torque exertion task, which has been widely used to control muscle activity, only the relevant joint torque is specified. However, the neglect of the neighboring joint could make the procedure unreliable, considering our previous result that even monoarticular muscle activity level is indefinite without specifying the adjacent joint torque. Here we examined the amount of hip joint torque generated with knee joint torque and its influence on the activity of the knee joint muscles. Twelve healthy subjects were requested to exert various levels of isometric knee joint torque. The knee and hip joint torques were obtained by using a custom-made device. Because no information about hip joint torque was provided to the subjects, the hip joint torque measured here was a secondary one associated with the task. The amount of hip joint torque varied among subjects, indicating that they adopted various strategies to achieve the task. In some subjects, there was a considerable internal variability in the hip joint torque. Such variability was not negligible, because the knee joint muscle activity level with respect to the knee joint torque, as quantified by surface electromyography (EMG), changed significantly when the subjects were requested to change the strategy. This change occurred in a very systematic manner: in the case of the knee extension, as the hip flexion torque was larger, the activity of mono- and biarticular knee extensors decreased and increased, respectively. These results indicate that the conventional single knee joint torque exertion has the drawback that the intersubject and/or intertrial variability is inevitable in the relative contribution among mono- and biarticular muscles because of the uncertainty of the hip joint torque. We discuss that the viewpoint that both joint torques need to be considered will bring insights into various controversial problems such as the shape of the EMG-force relationship, neural factors that help determine the effect of muscle strength training, and so on.