Phagenesis Ltd.

Aldolase C is regarded as the brain-specific form of fructose-1,6-bisphosphate aldolase whereas aldolase A is regarded as muscle-specific. In situ hybridization of mouse central nervous system using isozyme-specific probes revealed that aldolase A and C are expressed in complementary cell types. With the exception of cerebellar Purkinje cells, aldolase A mRNA is found in neurons; aldolase C message is detected in astrocytes, some cells of the pia mater, and Purkinje cells. We isolated aldolase C genomic clones that span the entire protein coding region from 1.5 kb 5 to the transcription start site to 0.5 kb 3 to the end of the last exon. The bacterial gene, lacZ, was inserted in two different locations and the constructs tested in transgenic mice. When the protein coding sequences were replaced with lacZ, three of five transgenic lines expressed ␤-galactosidase only in cells of the pia mater; one line also expressed in astrocyte-like cells. When lacZ was inserted into the final exon (and all structural gene sequences were retained) transgene expression was observed in astrocytes in all regions of the central nervous system as well as in pial cells. Thus, with the exception of Purkinje cell expression, the behavior of the full-length transgene mimics the endogenous aldolase C gene. The results with the shorter transgene suggest that additional enhancer elements exist within the intragenic sequences. The absence of Purkinje cell staining suggests that the cis elements required for this expression must be located outside of the sequences used in this study.

The qualitative relationship between preprotein translocases in the mitochondrial outer and inner membranes was determined by both a functional analysis and a determination of characteristic components of the translocases. Translocation contact sites of isolated mitochondria were saturated with intermediates of a matrix-targeted precursor of the ␤-subunit of the F 1 -ATPase (pF 1 ␤), and import of preproteins into the different mitochondrial subcompartments was monitored. A strong inhibition (75-95%) was observed for preproteins with an N-terminal matrix targeting signal, indicating that a significant portion of the contact sites was blocked by accumulated F 1 ␤. Insertion of preproteins into the outer membrane and import into the intermembrane space of preproteins without matrix targeting signals was inhibited by about 45%, indicating that functional outer membrane translocases were available despite saturation of contact sites. Similarly, import of members of the mitochondrial carrier family into the inner membrane was only partly inhibited (40 -50%), demonstrating that functional Tim22 translocases were available to cooperate with the Tom machinery in the import of carrier proteins. The stoichiometry of Tom40, Tim23, and Tim22 in mitochondria was determined to be 5:1:0.22. We conclude that translocases of the outer membrane are present in excess over translocases of the inner membrane. Import of nuclear encoded mitochondrial preproteins into the mitochondria is a multistep process (1). To reach the mitochondrial matrix, precursor proteins are translocated across the mitochondrial outer and inner membranes. Biochemical and morphological studies revealed that protein import into the matrix proceeds through proteinaceous channels at so-called translocation contact sites (2-7). In the electron microscope, regions of close proximity of the mitochondrial outer membrane to the inner membrane have been observed (5). These areas are particularly conspicuous in mitochondria in which the matrix space has been shrunk and the inner membrane is pulled away from the outer membrane. They are often referred to as morphological contact sites. Contact sites may be the preferred location where translocation contacts of the Tom and Tim complexes are formed, but this issue has not been finally settled. With biochemical means, contact sites between Tom and Tim complexes were only detected in the presence of a translocating polypeptide chain (6, 7). Components of the Tom machinery and Tim23, Tim17, as well as mt-Hsp70 and Tim44 were found in such super-complexes.For import of members of the mitochondrial carrier family, such as the ADP/ATP carrier, the Tom machinery cooperates with Tim22 (8). This pathway does not require functional Tim23 and mt-Hsp70 (8). The translocation machinery of the outer membrane can also act independent of the translocases of the inner membrane (9). Similarly, in mitoplast preparations, preproteins are imported directly via Tim23 into the matrix (10). This raises the question of how the Tom machinery inter...

Normal subjects use an open-loop motor control strategy in handwriting, but they are able to switch to closed-loop motor control when the demands on accuracy increase. These closed-loop handwriting movements of normal subjects resemble the inefficient movements found in writing-impaired patients. The hypothesis that such movement deficits may in fact reflect the use of a closed-loop strategy was tested in a group of writing-impaired patients with multiple sclerosis (MS). The handwriting movements of 10 MS patients and 20 control subjects were examined with a digitizing tablet. Three conditions were used: a standard writing task (expt 1), a closed-loop condition (expt 2), and an open-loop condition (expt 3). Individual stroke movements were analysed. Stroke duration and segmentation were increased for MS patients in the standard writing task. The same was found for control subjects when they wrote under closed-loop conditions. However, under open-loop conditions, the handwriting movements of the MS patients were as fast and fluent as that of control subjects. The results support the hypothesis that the movement characteristics of the writing-impaired MS patients reflect an inadequate use of a closed-loop motor control strategy.