The formation of actin rods composed of actin tubules in Dictyostelium discoideum spores.

A new type of actin rod formed in both the nucleus and the cytoplasm, as well as tyrosine phosphorylation of actin, is implicated in the maintenance of dormancy and viability of Dictyostelium discoideum spores. Here the ultrastructure of the rods and their relationship to the phosphorylation of actin were examined. The rods first appeared in premature spores at the midculmination stage as bundles composed of actin tubules hexagonally cross-linked. The 13-nm-diameter bundles were composed of three actin filaments. Formation of the actin rods begins during the late culmination stage and proceeds until 2 days after completion of fruiting bodies. The physical events occur in the following order; association of several modules of bundles, close packing and decrease in diameter of actin tubules, elongation of rods across the nucleus or the cytoplasm. Actin phosphorylation levels increased at the late culmination stage and reached a maximum level 12 h later. Immediately following activation of spore germination, actin was rapidly dephosphorylated, followed shortly thereafter by the disappearance of rods. Shortened actin tubules once again became arranged in a hexagonal pattern. This hexagonal arrangement of actin tubules is possibly involved in rod formation and disappearance and does not depend upon actin phosphorylation. In contrast, rod-maturation processes may correlate with actin phosphorylation.

Expression of brain-2 in the developing olfactory bulb.

The expression of Brain-2, a POU domain transcription factor, was examined in the developing olfactory bulb. Brain-2 was expressed mainly in the output neurons, mitral cell and tufted cells in the main olfactory bulb (MOB), and mitral/tufted cells (MT cells) in the accessory olfactory bulb (AOB). It was not expressed in granular cells in either the MOB or the AOB. Our results suggest that Brain-2 was specifically expressed in output neurons but not in interneurons in the developing olfactory bulb. Brain-2 may play a role in the development of these output neurons.

Predominant expression of Brn-2 in the postmitotic neurons of the developing mouse neocortex.

The expression of Brn-2, a central nervous systems (CNS)-specific POU domain transcription factor, in the developing mouse neocortex was examined with an anti-Brn-2 antibody. Brn-2 protein was first detected in CNS on embryonic day (E) 11.5, and remained strong until E15.5. From E11.5 to postnatal day (P) 0, a high level of Brn-2 expression was observed in the subventricular zone, the intermediate zone, and the outer layer of the neocortex, but not in the ventricular zone. In the double-staining experiments, most of the Brn-2 positive cells were also positive for NCAM-H, an adhesion molecule specific to post-mitotic neurons. Furthermore, BrdU-labeling experiments demonstrated the presence of Brn-2 protein exclusively in postmitotic cells. These results indicated that, in the developing neocortex, Brn-2 expression is up-regulated after the final cell division. Therefore, this transcription factor may be involved in the migration and/or maturation process of the immature neuronal cells.

Three-dimensional brain visualization for metachromatic leukodystrophy.

The basic understanding of many neurogenetic diseases requires study of the clinical, biochemical, and pathological aspects. To study the pathological aspects, the organs affected by the disease must be observed. We have used volume visualization techniques to create three-dimensional (3D) brain images of a patient with late infantile metachromatic leukodystrophy (MLD). The 3D brain images showed clearly, stereographically, and non-invasively the intracerebral lesion. This lesion, which indicated hyperintensity in magnetic resonance (MR) images, extended throughout the periventricular white matter. The 3D brain images are provided to integrate information. Volumetric ray-casting was useful in obtaining directly images of the entire brain and in allowing an intuitive understanding of the extension of the lesion in three dimensions and of the extent of the defects in the MLD brain. Isosurfacing facilitated a clear extraction of the lesion located by volumetric ray-casting. Each technique used in this study played a role in visualization and their use was complementary. 3D brain images will promote morphological investigation of neurogenetic diseases.

Quantitative analysis of dystrophin gene amplification products using a PC-based image analysis system.

A PC-based image analysis system for gel photographs of DNA gel patterns was developed. It was originally designed as a general-purpose, low-cost, yet high-performance system for wide applications in the biomedical area. In this study, we performed analysis of gel images obtained by polymerase chain reaction (PCR) amplification. The system employs a high-resolution CCD camera that can accurately measure grayness of the gel photographs for quantitative analysis of PCR products. The target DNA (exon 52 of the dystrophin gene), which had been found to be deleted in some patients with Duchenne/Becker muscular dystrophy (DMD/BMD), was amplified for the female family members together with the control DNA (exon 60) as a reference. The ratio of the target DNA to the control DNA was determined from PCR products to identify the carrier status of this disease by means of gene dosage. We conclude that 'the PC-based image analysis system' was useful for quantitative deletion analysis of DMD/BMD heterozygotes.

Carrier detection of Duchenne/Becker muscular dystrophy: computer-assisted direct quantitation of gene amplification products.

An improved method by quantitative dystrophin gene deletion analysis was developed for the detection of Duchenne/Becker muscular dystrophy (DMD/BMD) carriers. Exon 52, which had been found to be deleted in DMD probands, was amplified for female family members, together with exon 60 as a reference, at the exponential phase of polymerase chain reaction. The products were separated by electrophoresis, the band intensities on gel photographs were quantitated, and the target/control ratios were calculated. The values for three heterozygous mothers were approximately half those for normal individuals and two definite non-heterozygous mothers. This procedure is easy, rapid and useful for the carrier diagnosis of DMD/BMD.

Fabry disease: detection of 13-bp deletion in alpha-galactosidase A gene and its application to gene diagnosis of heterozygotes.

Polymerase chain reaction amplification of reverse-transcribed messenger RNA from a patient with Fabry disease revealed a 13-base pair deletion in the 5' region (exon 1) of alpha-galactosidase A complementary DNA. This gene rearrangement was not detected by Southern or Northern analysis. Short direct repeats were present around the breakpoints, and considered to be of pathogenetic significance. Gene diagnosis of the mother and a female cousin was successfully achieved by polymerase chain reaction amplification of genomic DNA; the former as a Fabry disease heterozygote and the latter as a normal homozygote.