Mesodermal and neuronal retinoids regulate the induction and maintenance of limb innervating spinal motor neurons

During embryonic development, the generation, diversification and maintenance of spinal motor neurons depend upon extrinsic signals that are tightly regulated. Retinoic acid (RA) is necessary for specifying the fates of forelimb-innervating motor neurons of the Lateral Motor Column (LMC), and the specification of LMC neurons into medial and lateral subtypes. Previous studies implicate motor neurons as the relevant source of RA for specifying lateral LMC fates at forelimb levels. However, at the time of LMC diversification, a significant amount of retinoids in the spinal cord originates from the adjacent paraxial mesoderm. Here we employ mouse genetics to show that RA derived from the paraxial mesoderm is required for lateral LMC induction at forelimb and hindlimb levels, demonstrating that mesodermally synthesized RA functions as a second source of signals to specify lateral LMC identity. Furthermore, reduced RA levels in postmitotic motor neurons result in a decrease of medial and lateral LMC neurons, and abnormal axonal projections in the limb; invoking additional roles for neuronally synthesized RA in motor neuron maintenance and survival. These findings suggest that during embryogenesis, mesodermal and neuronal retinoids act coordinately to establish and maintain appropriate cohorts of spinal motor neurons that innervate target muscles in the limb.     

Control of head stability during gait initiation in young and older women

Transition tasks between static and dynamic situations may challenge head stabilization and balance in older individuals. The study was designed to investigate differences between young and older women in the upper body motion during the voluntary task of gait initiation. Seven young (25 ± 2.3 years) and seven older healthy women (78 ± 3.4 years) were required to stand on a force platform and initiate walking at their self-selected preferred speed. Angles of head, neck and trunk were measured by motion analysis in the sagittal plane and a cross-correlation analysis was performed on segments pairs. Variability of head and neck angular displacements, as indicated by average standard deviation, was significantly greater in the older than in the young participants. The young women maintained dynamic stability of the upper body, as forward flexion of the trunk was consistently counteracted by coordinated head–neck extension. Differently, movement patterns employed by the older women also included a rigid motion of all upper body segments leaning forward as a single unit. These results demonstrated that older women perform the transition from standing to walking with greater variability in the patterns of upper body motion compared to young women.     

Colocalization studies of Arp1 and p150Glued to spindle microtubules during mitosis: the effect of cytochalasin on the organization of microtubules and motor proteins in PtK1 cells

Motor proteins play a fundamental role in the congression and segregation of chromosomes in mitosis as well as the formation of the mitotic spindle. In particular, the dynein/dynactin complex is involved in the maintenance of the spindle, formation of astral microtubules, chromosome motion, and chromosome segregation. Dynactin is a multisubunit, high molecular weight protein that is responsible for the attachment of cargo to dynein. There are a number of major subunits in dynactin that are presumed to be important during mitosis. Arp1 is thought to be the attachment site for cargo to the complex while p150(Glued), a side arm of this complex regulates binding to MTs and the binding of dynactin to dynein. We performed colocalization studies of Arp1 and p150(Glued) to spindle microtubules. Both Arp1 and p150(Glued) colocalize with spindle MTs as well as cytoplasmic components. When treated with cytochalasin J, Arp1 concentrates at the centrosomes and is less co-localized with spindle MTs. Cytochalasin J has less of an effect on the colocalization of p150(Glued) with spindle MTs, suggesting that Arp1 may have a cytochalasin J sensitive site.     

Use of Multiple-Site Performance-Contingent SEMG Reward Programming in Pediatric Rehabilitation: A Retrospective Review

We completed a retrospective review of the effectiveness of multi-site, performance-contingent reward programming on functional change in motor performance of 16 treatment resistant children. Patients were previously treated in physical or occupational therapy for head control, standing balance training, sitting and upper extremity use (brachial plexus injury). They then participated in a program that utilized multiple surface electromyography sites the use of which was rewarded with videos for performing the correct constellation of recruitment pattern (e.g., contracting some muscles while relaxing others). Onset of reward was calibrated for each patient and transfer of skill to outside the clinic was encouraged by linking a verbal cue to the correct motor plan. Fourteen of the 16 patients improved. The implications of the use of this technique in the treatment of motor dysfunction is discussed.     

Reconstitution of vacuolar-type rotary H+-ATPase/synthase from Thermus thermophilus

Vacuolar-type rotary H(+)-ATPase/synthase (V(o)V(1)) from Thermus thermophilus, composed of nine subunits, A, B, D, F, C, E, G, I, and L, has been reconstituted from individually isolated V(1) (A(3)B(3)D(1)F(1)) and V(o) (C(1)E(2)G(2)I(1)L(12)) subcomplexes in vitro. A(3)B(3)D and A(3)B(3) also reconstituted with V(o), resulting in a holoenzyme-like complexes. However, A(3)B(3)D-V(o) and A(3)B(3)-V(o) did not show ATP synthesis and dicyclohexylcarbodiimide-sensitive ATPase activity. The reconstitution process was monitored in real time by fluorescence resonance energy transfer (FRET) between an acceptor dye attached to subunit F or D in V(1) or A(3)B(3)D and a donor dye attached to subunit C in V(o). The estimated dissociation constants K(d) for V(o)V(1) and A(3)B(3)D-V(o) were ～0.3 and ～1 nm at 25 °C, respectively. These results suggest that the A(3)B(3) domain tightly associated with the two EG peripheral stalks of V(o), even in the absence of the central shaft subunits. In addition, F subunit is essential for coupling of ATP hydrolysis and proton translocation and has a key role in the stability of whole complex. However, the contribution of the F subunit to the association of A(3)B(3) with V(o) is much lower than that of the EG peripheral stalks.     

Airborne hyperspectral imaging for estimating acorn yield based on the PLS B-matrix calibration technique

Alternate bearing of acorn is a well-marked yield variability phenomenon in forest production. In Japan, this phenomenon is also related to wildlife management (e.g. of animals such as wild pigs, that rely on acorn as their major feed source). Effective management of animals dependent on acorn will require accurate estimation of acorn yield at an early stage. In this paper, we proposed a way to estimate acorn yield from the canopy reflectance values of individual trees. Using an Airborne Imaging Spectrometer for Application (AISA) Eagle System, hyperspectral images in 72 visible and near-infrared wavelengths (407–898 nm) were acquired over an acorn forest in Japan 10 times over three consecutive years (2003–2005) during the early acorn growing season. The canopy spectral reflectance values for individual trees at each wavelength were extracted from the images, and important wavelengths were determined as estimating factors by the B-matrix technique based on partial least squares (PLS) analysis. Yield-estimating models were then developed by multiple linear regression (MLR). Three models obtained from images acquired on June 27 in 2003, July 13 in 2004 and June 21 in 2005 estimated acorn yield well in comparison with ground truth, indicating that the procedure has considerable potential. The study also demonstrated the B-matrix technique based on PLS analysis to be reliable and efficient in identifying important wavelengths for determining suitable estimating factors that best contribute to the estimation model.     

Targeted disruption of the Pak5 and Pak6 genes in mice leads to deficits in learning and locomotion

PAK6 is a member of the group B family of PAK serine/threonine kinases, and is highly expressed in the brain. The group B PAKs, including PAK4, PAK5, and PAK6, were first identified as effector proteins for the Rho GTPase Cdc42. They have important roles in filopodia formation, the extension of neurons, and cell survival. Pak4 knockout mice die in utero, and the embryos have several abnormalities, including a defect in the development of motor neurons. In contrast, Pak5 knockout mice do not have any noticeable abnormalities. So far nothing is known about the biological function of Pak6. To address this, we have deleted the Pak6 gene in mice. Since Pak6 and Pak5 are both expressed in the brain, we also generated Pak5/Pak6 double knockout mice. These mice were viable and fertile, but had several locomotor and behavioral deficits. Our results indicate that Pak5 and Pak6 together are not required for viability, but are required for a normal level of locomotion and activity as well as for learning and memory. This is consistent with a role for the group B PAKs in the nervous system.     

Intracellular imaging of targeted proteins labeled with quantum dots

We developed a new method for imaging the movement of targeted proteins in living cancer cells with photostable and bright quantum dots (QDs). QDs were conjugated with various molecules and proteins, such as phalloidin, anti-tubulin antibody and kinesin. These bioconjugated QDs were mixed with a transfection reagent and successfully internalized into living cells. The movements of individual QDs were tracked for long periods of time. Phalloidin conjugated QDs bound to actin filaments and showed almost no movement. In contrast, anti-tubulin antibody conjugated QDs bound to microtubules and revealed dynamic movement of microtubules. Kinesin showed an interesting behavior whereby kinesin came to be almost paused briefly for a few seconds and then moved once again. This is in direct contrast to the smoothly continuous movement of kinesin in an in vitro assay. The maximum velocity of kinesin in cells was faster than that in the in vitro assay. These results suggest that intracellular movement of kinesin is different from that in the in vitro assay. This newly described method will be a powerful tool for investigating the functions of proteins in living cells.     

Enhanced motor unit rate coding with improvements in a force-matching task

These data describe improved modulation of discharge rates (rate coding) of first dorsal interosseous motor units throughout the acquisition of a complex force-matching skill involving isometric index finger abduction. In each of 15 consecutive trials, subjects attempted to match their force to a trajectory consisting of the sum of two sine waves (0.15 and 0.5 Hz) and random oscillations (overall mean force level ˜20% MVC). Reductions in root-mean-square (RMS) error of each subject’s force relative to the trajectory indicated substantial improvements in force-matching ability (F=33.8, p<0.001). With the acquisition of this new skill, there was increased amplitude modulation of muscular force near both dominant frequencies of the force-matching trajectory (F=10.6, p=0.008). The standard deviation and coefficient of variation of motor unit inter-spike intervals both decreased with improved performance indicating a general reduction in the amplitude of firing rate modulations (SD: F=18.69, p=0.001; CV: F=43.6, p<0.001). After skill acquisition, there was decreased firing rate modulation outside of the two dominant frequencies and increased amplitude of firing rate modulation at the higher of the two dominant frequencies (0.5 Hz, F=8.23, p=0.015). These findings indicate that improved precision of rate coding was a contributor to the acquisition of the new force-matching task. That the change in rate coding was frequency dependent suggests that factors other than frequency coding may contribute to the improved force matching at 0.15 Hz.     

The kinesin I family member KIF5C is a novel substrate for protein kinase CK2

Protein kinase CK2 is ubiquitously expressed. The holoenzyme is composed of two catalytic α- or α′-subunits and two regulatory β-subunits but evidence is accumulating that the subunits can function independently. The composition of the holoenzyme as well as the expression of the individual subunits varies in different tissues, with high expression of CK2α′ in testis and brain. CK2 phosphorylates a number of different substrates which are implicated in basal cellular processes such as proliferation and survival of cells. Here, we report a new substrate, KIF5C, which is a member of the kinesin 1 family of motor neuron proteins. Phosphorylation of KIF5C was demonstrated in vitro and in vivo. Using deletion mutants, a peptide library, and mutation analysis a phosphorylation site for CK2 was mapped to amino acid 338 which is located in the non-motor domain of KIF5C. Interestingly, KIF5C is phosphorylated by holoenzymes composed of CK2α/CK2β and CK2α′/CK2β as well as by CK2α′ alone but not by CK2α alone.