Axon diameters and conduction velocities in the macaque pyramidal tract.

Small axons far outnumber larger fibers in the corticospinal tract, but the function of these small axons remains poorly understood. This is because they are difficult to identify, and therefore their physiology remains obscure. To assess the extent of the mismatch between anatomic and physiological measures, we compared conduction time and velocity in a large number of macaque corticospinal neurons with the distribution of axon diameters at the level of the medullary pyramid, using both light and electron microscopy. At the electron microscopic level, a total of 4,172 axons were sampled from 2 adult male macaque monkeys. We confirmed that there were virtually no unmyelinated fibers in the pyramidal tract. About 14% of pyramidal tract axons had a diameter smaller than 0.50 µm (including myelin sheath), most of these remaining undetected using light microscopy, and 52% were smaller than 1 µm. In the electrophysiological study, we determined the distribution of antidromic latencies of pyramidal tract neurons, recorded in primary motor cortex, ventral premotor cortex, and supplementary motor area and identified by pyramidal tract stimulation (799 pyramidal tract neurons, 7 adult awake macaques) or orthodromically from corticospinal axons recorded at the mid-cervical spinal level (192 axons, 5 adult anesthetized macaques). The distribution of antidromic and orthodromic latencies of corticospinal neurons was strongly biased toward those with large, fast-conducting axons. Axons smaller than 3 µm and with a conduction velocity below 18 m/s were grossly underrepresented in our electrophysiological recordings, and those below 1 µm (6 m/s) were probably not represented at all. The identity, location, and function of the majority of corticospinal neurons with small, slowly conducting axons remains unknown.

Measures of spinal canal stenosis and relationship to spinal cord structure in patients with cervical spondylosis.

OBJECTIVES: Spinal canal stenosis is often measured on anatomical magnetic resonance imaging (MRI) to estimate the degree of spinal cord compression. This study examined whether two quantitative measures of spinal canal stenosis taken from anatomical MRI are related to spinal cord white-matter integrity in patients with cervical spondylosis measured by diffusion tensor imaging (DTI). PATIENTS AND METHODS: DTI and T2-weighted MRI of the cervical spinal cord were performed in 15 patients with cervical spondylosis and ten healthy control subjects of similar age. Severity of stenosis was calculated using Pavlov's ratio and the space-available-for-cord (SAC) technique. RESULTS: Patients had significantly lower Pavlov's ratios and SAC (C2-C3, C4-C5 and C6-C7), lower fractional anisotropy (FA; C2-C3 and C4-C5) and higher radial diffusivity (C2-C3, C4-C5 and C6-C7) than the controls. In patients, only Pavlov's ratio correlated with mean FA (R=0.66, P=0.008). Variations in Pavlov's ratio and FA also showed a similar pattern across cervical levels. CONCLUSION: Pavlov's ratio is a better predictor of spinal cord integrity than the SAC and, therefore, may be more relevant clinically for the evaluation of stenosis in patients with cervical spondylosis.

Impact of precision grip tasks on cervical spinal network excitability in humans.

Motor skill acquisition in the lower limb may induce modifications of spinal network excitability. We hypothesized that short-term motor adaptation in precision grip tasks would also induce modifications of cervical spinal network excitability. In a first series of experiments, we studied the impact of two different precision grip force control tasks (a visuomotor force-tracking task and a control force task without visual feedback) on cervical spinal network excitability in healthy subjects. We separately tested the efficacy of two key components of the spinal circuitry: (i) presynaptic inhibition on flexor carpi radialis (FCR) Ia terminals, and (ii) disynaptic inhibition directed from extensor carpi radialis (ECR) to FCR. We found that disynaptic inhibition decreased temporarily after both force control tasks, independently of the presence of visual feedback. In contrast, the amount of presynaptic inhibition on FCR Ia terminals decreased only after the visuomotor force tracking task. This temporary decrease was correlated with improved tracking accuracy during the task (i.e. short-term motor adaptation). A second series of experiments confirmed these results and showed that the visuomotor force-tracking task resulted also in an increase of the Hmax/Mmax ratio and the slope of the ascending part of the H-reflex recruitment curve. In order to address the role of presynaptic inhibition in the motor adaptation process, we conducted a third series of experiments during which presynaptic inhibition was recorded before and after two consecutive sessions of visuomotor force tracking. The results showed that (i) improved tracking accuracy occurred during both sessions, and (ii) presynaptic inhibition decreased only after the first session of visuomotor force tracking. Taken together, these results suggest thus that the nature of the motor task performed has a specific impact on the excitability of these cervical spinal circuits. These findings also suggest that early motor adaptation is associated with a modulation of presynaptic Ia inhibition in the upper limb.

Dynamic neural network models of the premotoneuronal circuitry controlling wrist movements in primates.

Dynamic recurrent neural networks were derived to simulate neuronal populations generating bidirectional wrist movements in the monkey. The models incorporate anatomical connections of cortical and rubral neurons, muscle afferents, segmental interneurons and motoneurons; they also incorporate the response profiles of four populations of neurons observed in behaving monkeys. The networks were derived by gradient descent algorithms to generate the eight characteristic patterns of motor unit activations observed during alternating flexion-extension wrist movements. The resulting model generated the appropriate input-output transforms and developed connection strengths resembling those in physiological pathways. We found that this network could be further trained to simulate additional tasks, such as experimentally observed reflex responses to limb perturbations that stretched or shortened the active muscles, and scaling of response amplitudes in proportion to inputs. In the final comprehensive network, motor units are driven by the combined activity of cortical, rubral, spinal and afferent units during step tracking and perturbations. The model displayed many emergent properties corresponding to physiological characteristics. The resulting neural network provides a working model of premotoneuronal circuitry and elucidates the neural mechanisms controlling motoneuron activity. It also predicts several features to be experimentally tested, for example the consequences of eliminating inhibitory connections in cortex and red nucleus. It also reveals that co-contraction can be achieved by simultaneous activation of the flexor and extensor circuits without invoking features specific to co-contraction.

Kinematic and dynamic synergies of human precision-grip movements.

We analyzed the adaptability of human thumb and index finger movement kinematics and dynamics to variations of precision grip aperture and movement velocity. Six subjects performed precision grip opening and closing movements under different conditions of movement velocity and movement aperture (thumb and index finger tip-to-tip distance). Angular motion of the thumb and index finger joints was recorded with a CyberGlove and a three-dimensional biomechanical model was used for solving the inverse dynamics problem during precision grip movements, i.e., for calculating joint torques from experimentally obtained angular variations. The time-varying joint angles and joint torques were analyzed by principal-component analysis to quantify the contributions of individual joints in kinematic and dynamic synergies. At the level of movement kinematics, we found subject-specific angular contributions. However, the adaptation to large aperture, achieved by an increase of the relative contribution of the proximal joints, was subject-invariant. At the level of movement dynamics, the adaptation of thumb-index finger movements to task constraints was similar among all subjects and required the linear scaling of joint torques, the synchronization of joint torques under high velocity conditions, and a flexible redistribution of joint torques between the proximal joint of the thumb and that of the index finger. This work represents one of the first attempts at calculating the joint torques during human precision-grip movements and indicates that the dynamic synergies seem to be remarkably simple compared with the synergies found for movement kinematics.

Macaque ventral premotor cortex exerts powerful facilitation of motor cortex outputs to upper limb motoneurons.

The ventral premotor area (F5) is part of the cortical circuit controlling visuomotor grasp. F5 could influence hand motor function through at least two pathways: corticospinal projections and corticocortical projections to primary motor cortex (M1). We found that stimulation of macaque F5, which by itself evoked little or no detectable corticospinal output, could produce a robust modulation of motor outputs from M1. Arrays of fine microwires were implanted in F5 and M1. During terminal experiments under chloralose anesthesia, single stimuli delivered to M1 electrodes evoked direct (D) and indirect (I1,I2, and I3) corticospinal volleys. In contrast, single F5 shocks were ineffective; double shocks (3 msec separation) evoked small I waves but no D wave. However, when the test (T) M1 shock was conditioned (C) by single or double F5 shocks, there was strong facilitation of I2 and I3 waves from M1, with C-T intervals of <1 msec. Intracellular recordings from 79 arm and hand motoneurons (MNs) revealed no postsynaptic effects from single F5 shocks. In contrast, these stimuli produced a robust facilitation of I2 and I3 EPSPs evoked from M1 (60% of MNs); this was particularly marked in hand muscle MNs (92%). Muscimol injection in M1 reduced I waves from F5 and abolished the F5-induced facilitation of late I waves from M1, and of EPSPs associated with them. Thus, some motor effects evoked from F5 may be mediated by corticocortical inputs to M1 impinging on interneurons generating late corticospinal I waves. Similar mechanisms may allow F5 to modulate grasp-related outputs from M1.

Facilitation from ventral premotor cortex of primary motor cortex outputs to macaque hand muscles.

We demonstrate that in the macaque monkey there is robust, short-latency facilitation by ventral premotor cortex (area F5) of motor outputs from primary motor cortex (M1) to contralateral intrinsic hand muscles. Experiments were carried out on two adult macaques under light sedation (ketamine plus medetomidine HCl). Facilitation of hand muscle electromyograms (EMG) was tested using arrays of fine intracortical microwires implanted, respectively, in the wrist/digit motor representations of F5 and M1, which were identified by previous mapping with intracortical microstimulation. Single pulses (70-200 microA) delivered to F5 microwires never evoked any EMG responses, but small responses were occasionally seen with double pulses (interval: 3 ms) at high intensity. However, both single- and double-pulse stimulation of F5 could facilitate the EMG responses evoked from M1 by single shocks. The facilitation was large (up to 4-fold with single and 12-fold with double F5 shocks) and occurred with an early onset, with significant effects at intervals of only 1-2 ms between conditioning F5 and test M1 stimuli. A number of possible pathways could be responsible for these effects, although it is argued that the most likely mechanism would be the facilitation, by cortico-cortical inputs from F5, of corticospinal I wave activity evoked from M1. This facilitatory action could be of considerable importance for the coupling of grasp-related neurons in F5 and M1 during visuomotor tasks.

Interspecies comparisons for the C3-C4 propriospinal system: unresolved issues.

There are conflicting views on the functional importance of the system of C3-C4 propriospinal neurones in the macaque, although the two sets of observations from the opposing laboratories are actually quite similar, both making the system appear much weaker than its well-known equivalent in the cat. One group asserts, mainly via evidence derived from experiments with strychnine, that this is a consequence simply of inhibition of the propriospinal neurone. However, we ague here that this judgement is premature and that much more needs to be known about the neurones involved and their connectivity before the analogy with the system in the cat is safe. This is particularly important because of a similar analogy which has been made with respect to measurements in human subjects.

Functional differences in corticospinal projections from macaque primary motor cortex and supplementary motor area.

We made a quantitative comparison of the density of macaque corticospinal projections from primary motor cortex (M1) and supplementary motor area (SMA) to spinal motor nuclei supplying hand and finger muscles. We also compared the action of corticospinal outputs from these two areas on 84 upper limb (mostly hand) motoneurones in chloralose-anaesthetised macaques. The hand representations of M1 and SMA were first identified using MRI and intracortical microstimulation. We made focal injections of WGA-HRP into these representations. Densitometric analysis showed that corticospinal projections from M1 were far denser and occupied a much greater proportion of the hand muscle motor nuclei than did SMA projections. Stimulation of M1 and SMA with bipolar intracortical pulses evoked monosynaptic EPSPs. These were significantly larger and more common from M1 (88% of motoneurons) than from SMA (48%). The results demonstrate corticomotoneuronal connections from both M1 and SMA, some converging upon single motoneurons. Both areas give rise to CM projections but that those from M1 are far more numerous and exert stronger excitatory effects than those from the SMA.

Longitudinal study of motor recovery after stroke: recruitment and focusing of brain activation.

BACKGROUND AND PURPOSE: The goal of this study was to characterize cortical reorganization after stroke and its relation with the site of the stroke-induced lesion and degree of motor recovery using functional MRI (fMRI). METHODS: Fourteen stroke patients with an affected upper limb were studied longitudinally. Three fMRI sessions were performed over a period of 1 to 6 months after stroke. Upper limb recovery, Wallerian degeneration of the pyramidal tract, and responses to transcranial magnetic stimulation were assessed. RESULTS: Two main patterns of cortical reorganization were found. Pattern 1 was focusing, in which, after initial recruitment of additional ipsilateral and contralateral areas, activation gradually developed toward a pattern of activation restricted to the contralateral sensorimotor cortex in 9 patients. Five patients were found to have pattern 2, persistent recruitment, in which there was an initial and sustained recruitment of ipsilateral activity. Occurrence of recruitment or focusing seemed to depend mainly on whether the primary motor cortex (M1) was lesioned; persistent recruitment was observed in 3 of 4 patients with M1 injury, and focusing was seen in 8 of 10 patients with spared M1. These patterns had no relation to the degree of recovery; in particular, focusing did not imply recovery. However, there was a clear relation between the degree of recovery and the degree of Wallerian degeneration. CONCLUSIONS: These results suggest that ipsilateral recruitment after stroke corresponds to a compensatory corticocortical process related to the lesion of the contralateral M1 and that the process of compensatory recruitment will persist if M1 is lesioned; otherwise, it will be transient.

Differences in the corticospinal projection from primary motor cortex and supplementary motor area to macaque upper limb motoneurons: an anatomical and electrophysiological study.

To further our understanding of the functional roles of different motor cortical areas, we made a quantitative comparison of the density of corticospinal projections from primary motor cortex (M1) and supplementary motor area (SMA) to spinal motor nuclei supplying hand and finger muscles in four macaque monkeys. We also compared the action of corticospinal outputs excited by electrical stimulation of these two areas on upper limb motoneurons recorded in three anaesthetized macaques. The hand representations of SMA and M1 were first identified using structural magnetic resonance imaging scans and intracortical microstimulation. In the anatomical study we then made focal injections of wheatgerm agglutinin- horseradish peroxidase into these representations, which were subsequently confirmed by analysis of retrograde cortical labelling. Densitometric analysis showed that corticospinal projections from M1 were denser and occupied a greater proportion of the hand muscle motor nuclei than did projections from SMA. In caudal Th1 the densest projections from M1 occupied 81% of this motoneuronal area, compared with only 6% from SMA. In the electrophysiological study, bipolar intracortical stimulation of the hand representation of M1 and SMA evoked direct (D) and indirect (I) corticospinal volleys. Volleys elicited by M1 stimulation had larger amplitudes and faster conduction velocities than those evoked from the SMA. Intracellular recordings were made from 84 contralateral upper limb motoneurons. M1 and SMA stimulation evoked markedly different responses in tested motoneurons: EPSPs were larger and more common from M1 (88% of motoneurons) than from SMA (48%). Some motoneurons (16/84) showed evidence of excitatory postsynaptic potentials mediated by monosynaptic action of the D-wave evoked from M1; these early effects were not observed from the SMA. In most motoneurons (74/84) EPSPs had segmental latencies indicating that they were due to monosynaptic action of the I-wave. The results are consistent with cortico-motoneuronal (CM) connections originating from both SMA and M1 converging upon single motoneurons, but those from M1 are far more numerous and exert stronger excitatory effects than from the SMA. Thus although they may function in parallel, the two CM projections probably make different contributions to upper limb motor control.

Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogs.

The antisense activity of oligomers with 2'-O-methyl (2'-O-Me) phosphorothioate, 2'-O-methoxyethyl (2'-O-MOE) phosphorothioate, morpholino and peptide nucleic acid (PNA) backbones was investigated using a splicing assay in which the modified oligonucleotides blocked aberrant and restored correct splicing of modified enhanced green fluorescent protein (EGFP) precursor to mRNA (pre-mRNA), generating properly translated EGFP. In this approach, antisense activity of each oligomer was directly proportional to up-regulation of the EGFP reporter. This provided a positive, quantitative readout for sequence-specific antisense effects of the oligomers in the nuclei of individual cells. Nuclear localization of fluorescent labeled oligomers confirmed validity of the functional assay. The results showed that the free uptake and the antisense efficacy of neutral morpholino derivatives and cationic PNA were much higher than that of negatively charged 2'-O-Me and 2'-O-MOE congeners. The effects of the PNA oligomers were observed to be dependent on the number of L-lysine (Lys) residues at the C-terminus. The experiments suggest that the PNA containing Lys was taken up by a mechanism similar to that of cell-penetrating homeodomain proteins and that the Lys tail enhanced intracellular accumulation of PNA oligomer without affecting its ability to reach and hybridize to the target sequence.

Peptide nucleic acids are potent modulators of endogenous pre-mRNA splicing of the murine interleukin-5 receptor-alpha chain.

Antisense oligonucleotides (ASOs) that bind target pre-mRNA with high affinity have been shown to alter splicing patterns and offer promise as therapeutics. Previous studies have shown that ASOs fully modified with 2'-O-methoxyethyl (2'-O-MOE) sugar residues redirect constitutive and alternative splicing of the murine interleukin-5 receptor-alpha (IL-5Ralpha) chain pre-mRNA in cells, resulting in inhibition of the membrane-bound isoform and enhanced expression of the soluble isoform. Here, we show that antisense peptide nucleic acids (PNAs) alter splicing of the IL-5Ralpha pre-mRNA in a fashion similar to their 2'-O-MOE-modified counterparts of the same sequence. Moreover, using PNA as the splicing modulator, the length of the antisense oligomer could be shortened from 20 to 15 nucleobase units to obtain a comparable effect. Treatment of cells with antisense PNA resulted in dose-dependent, specific downregulation of IL-5Ralpha membrane isoform mRNA expression and enhanced levels of the soluble IL-5Ralpha isoform transcript, with an EC50 equivalent to that observed in parallel with the corresponding 2'-O-MOE ASO. The pronounced activity of antisense PNAs in modulating IL-5Ralpha mRNA splicing observed in our study identifies these compounds as a promising new class of lower molecular weight splicing modulators.

Synthesis of novel 3'-C-methylene thymidine and 5-methyluridine/cytidine H-phosphonates and phosphonamidites for new backbone modification of oligonucleotides.

Novel 5'-O-DMT- and MMT-protected 3'-C-methylene-modified thymidine, 5-methyluridine, and 5-methylcytidine H-phosphonates 1-7 with O-methyl, fluoro, hydrogen, and O-(2-methoxyethyl) substituents at the 2'-position have been synthesized by a new effective strategy from the corresponding key intermediates 3'-C-iodomethyl nucleosides and intermediate BTSP, prepared in situ through the Arbuzov reaction. The modified reaction conditions for the Arbuzov reaction prevented the loss of DMT- and MMT-protecting groups, and directly provided the desired 5'-O-DMT- and/or MMT-protected 3'-C-methylene-modified H-phosphonates 1-6 although some of them were also prepared through the manipulation of protecting groups after the P-C bond formation. The modified Arbuzov reaction of 3'-C-iodomethyl-5-methylcytidine 53, prepared from its 5-methyluridine derivative 42, with BTSP provided the 5-methylcytidine H-phosphonate 54, which was further transferred to the corresponding 4-N-(N-methylpyrrolidin-2-ylidene)-protected H-phosphonate monomer 7. 5'-O-MMT-protected 3'-C-methylene-modified H-phosphonates 5, 3, and 7 were converted to the corresponding cyanoethyl H-phosphonates 50, 51, and 56 using DCC as a coupling reagent. One-pot three-step reactions of 50, 51, and 56 provided the desired 3'-C-methylene-modified phosphonamidite monomers 8-10. Some of these new 3'-methylene-modified monomers 1-10 have been successfully utilized for the synthesis of 3'-methylene-modified oligonucleotides, which have shown superior antisense properties including nuclease resistance and binding affinity to the target RNA.

Detection of alkali metal ions in DNA crystals using state-of-the-art X-ray diffraction experiments.

The observation of light metal ions in nucleic acids crystals is generally a fortuitous event. Sodium ions in particular are notoriously difficult to detect because their X-ray scattering contributions are virtually identical to those of water and Na(+.)O distances are only slightly shorter than strong hydrogen bonds between well-ordered water molecules. We demonstrate here that replacement of Na(+) by K(+), Rb(+) or Cs(+) and precise measurements of anomalous differences in intensities provide a particularly sensitive method for detecting alkali metal ion-binding sites in nucleic acid crystals. Not only can alkali metal ions be readily located in such structures, but the presence of Rb(+) or Cs(+) also allows structure determination by the single wavelength anomalous diffraction technique. Besides allowing identification of high occupancy binding sites, the combination of high resolution and anomalous diffraction data established here can also pinpoint binding sites that feature only partial occupancy. Conversely, high resolution of the data alone does not necessarily allow differentiation between water and partially ordered metal ions, as demonstrated with the crystal structure of a DNA duplex determined to a resolution of 0.6 A.

EMG activation patterns during force production in precision grip. III. Synchronisation of single motor units.

Motor unit (MU) synchronisation during isometric force production in the precision grip was analysed in five subjects performing a visually guided steptracking motor task with three different force levels. With this aim multi-unit electromyographic (EMG) activity of 14 intrinsic and extrinsic finger muscles from 15 experimental sessions was decomposed into the potentials of single MUs. The behaviour of 62 intrinsic and 30 extrinsic MUs in the motor task was quantified. Most MUs displayed a positive correlation between firing rate and grip force. Compared to MUs in extrinsic muscles, intrinsic MUs had steeper regression lines with negative intercepts indicating higher force sensitivity and higher recruitment thresholds. A cross-correlation analysis was performed for 69 intra- and 166 intermuscular MU pairs while steady grip force was exerted at the three force levels. Synchronisation, for at least one force level, was found in 78% of the intra- and 45% of the intermuscular pairs. The occurrence of synchronisation was not stable over the force range tested. Factors influencing the fluctuations in occurrence and strength of synchronisation were investigated. Force increase was not paralleled by increased synchronisation; in contrast, in most MU pairs, especially intermuscular pairs, synchronisation occurred preferentially at the lower force levels. The recruitment threshold appeared to play a determining role in synchronisation: the more similar the thresholds of two MUs, the greater the probability of them being synchronised at this force level. Synchronised MUs fired on average at a lower frequency than non-synchronised ones. Finally, synchronisation at the multi-unit EMG level does not indicate that all underlying MUs are synchronised, nor does the absence of temporal coupling at the multi-unit level indicate that none of the MUs is synchronised.

Striking differences in transmission of corticospinal excitation to upper limb motoneurons in two primate species.

There is considerable debate as to the relative importance, for cortical control of upper limb movements, of direct cortico-motoneuronal (CM) versus indirect, propriospinal transmission of corticospinal excitation to cervical motoneurons. In the cat, which has no CM connections, a significant proportion of corticospinal excitation reaches forelimb motoneurons via a system of C(3)-C(4) propriospinal neurons (PN). In contrast, in the macaque monkey most motoneurons receive direct CM connections, and, under the same experimental conditions as in the cat, there is little evidence for PN transmission. We have investigated corticospinal transmission in the New World squirrel monkey (Saimiri sciureus) because its CM projections are weaker than in the macaque. Intracellular recordings were made from motoneurons identified from the ulnar, median, and deep radial (DR) nerves in four adult squirrel monkeys under chloralose anesthesia and neuromuscular paralysis. Responses to stimulation of the contralateral medullary pyramid were recorded before and after a lesion to the dorsolateral funiculus (DLF) at C(5), designed to interrupt direct corticospinal inputs to the lower cervical segments and unmask PN-mediated effects. This lesion greatly reduced the proportion of motoneurons showing either CM EPSPs or disynaptic IPSPs, but the proportion showing late EPSPs with segmental latencies beyond the monosynaptic range, evoked by repetitive but not single PT stimuli, was unaffected: 23 of 29 motoneurons (79%) before and 32 of 37 (86%) after the lesion; 41% of these late EPSPs had strictly disynaptic latencies after the lesion, only 14% before. These results are in striking contrast to the macaque (late EPSPs in only 18% of motoneurons before a C(5) lesion, 19% after it). Transmission of the late EPSPs via C(3)-C(4) PNs in the squirrel monkey was indicated by their absence after an additional C(2) DLF lesion. Nearly all tested motoneurons also responded with short latency EPSPs to stimulation in the ipsilateral lateral reticular nucleus. By analogy with the cat, these EPSPs probably reflect antidromic activation of ascending collaterals of C(3)-C(4) PNs with monosynaptic connections to motoneurons; the EPSPs were significantly smaller than in the cat but larger than in the macaque. These results suggest that the positive correlation across species between more advanced hand function and the strength of the CM system is accompanied by a negative correlation between hand function and the strength of the PN system. We hypothesize that in primates with more advanced hand function, the CM system effectively replaces PN-mediated control. This would include a contribution to the control of reaching movements, which are said to be specifically under the control of the PN system in the cat, and we speculate that these differences may be related to the degree of dexterity exhibited by the different species. This interpretation of the results predicts that in man, where the CM system is highly developed, the PN system is unlikely to be responsible for significant transmission of cortical commands to upper limb motoneurons.

Synthesis of chimeric oligonucleotides containing phosphodiester, phosphorothioate, and phosphoramidate linkages.

[reaction: see text] H-Phosphonate monomers of 2'-O-(2-methoxyethyl) ribonucleosides have been synthesized. Oxidation of oligonucleotide H-phosphonates has been optimized to allow the synthesis of oligonucleotides containing either 2'-deoxy or 2'-O-(2-methoxyethyl) ribonucleoside residues combined with three different phosphate modifications in the backbone, i.e., phosphodiester (PO), phosphorothioate (PS), and phosphoramidate (PN). Phosphodiester linkages were introduced by oxidation with a cocktail of 0.1 M Et(3)N in CCl(4)/Pyr/H(2)O (5:9:1) without affecting phosphorothioate or phosphoramidate linkages. For the synthesis of phosphoramidate-modified oligonucleotides, N(4)-acetyl deoxycytidine-3'-H-phosphonate monomers were used to avoid transamination during the oxidation step.

Aromatic hydrocarbon receptor interaction with the retinoblastoma protein potentiates repression of E2F-dependent transcription and cell cycle arrest.

Polyhalogenated aromatic hydrocarbons, of which 2,3,7, 8-tetrachloro-p-dioxin (TCDD) is the prototype compound, elicit a variety of toxic, teratogenic, and carcinogenic responses in exposed animals and in humans. In cultured cells, TCDD shows marked effects on the regulation of cell cycle progression, including thymocyte apoptosis, induction of keratinocyte proliferation and terminal differentiation, and inhibition of estrogen-dependent proliferation in breast cancer cells. The presence of an LXCXE domain in the dioxin aromatic hydrocarbon receptor (AHR), suggested that the effects of TCDD on cell cycle regulation might be mediated by protein-protein interactions between AHR and the retinoblastoma protein (RB). Using the yeast two-hybrid system, AHR and RB were in fact shown to bind to each other. In vitro pull-down experiments with truncated AHR peptides indicated that at least two separate AHR domains form independent complexes with hypophosphorylated RB. Coimmunoprecipitation of whole cell lysates from human breast carcinoma MCF-7 cells, which express both proteins endogenously, revealed that AHR associates with RB in vivo only after receptor transformation and nuclear translocation. However, the AHR nuclear translocator and transcriptional heterodimerization partner, is not required for (nor is it a part of) the AHR.RB complexes detected in vitro. Ectopic expression of AHR and RB in human osteosarcoma SAOS-2 cells, which lack endogenous expression of both proteins, showed that AHR synergizes with RB to repress E2F-dependent transcription and to induce cell cycle arrest. Furthermore, AHR partly blocked T-antigen-mediated reversal of RB-dependent transcriptional repression. These results uncover a potential function for the AHR in cell cycle regulation and suggest that this function may be that of serving as an environmental sensor that signals cell cycle arrest when cells are exposed to certain environmental toxicants.