An acute dose of gamma-hydroxybutyric acid alters gene expression in multiple mouse brain regions.

Gamma-hydroxybutyric acid (GHB) is normally found in the brain in low concentrations and may function as a neurotransmitter, although the mechanism of action has not been completely elucidated. GHB has been used as a general anesthetic and is currently used to treat narcolepsy and alcoholism. Recreational use of GHB is primarily as a "club drug" and a "date rape drug," due to its amnesic effects. For this study, the hypothesis was that behavioral and neurochemical alterations may parallel gene expression changes in the brain after GHB administration. Adult male C57/B6N mice (n=5/group) were administered a single dose of 500 mg/kg GHB (i.p.) and were sacrificed 1, 2 and 4 h after treatment. Control mice were administered saline. Brains were removed and regionally dissected on ice. Total RNA from the hippocampus, cortex and striatum was extracted, amplified and labeled. Gene expression was evaluated using Agilent whole mouse genome 4x44K oligonucleotide microarrays. Microarray data were analyzed by ArrayTrack and differentially expressed genes (DEGs) were identified using P < or = 0.01 and a fold change > or = 1.7 as the criteria for significance. Principal component analysis (PCA) and Hierarchical Cluster Analysis (HCA) showed that samples from each time point clustered into distinct treatment groups with respect to sacrifice time. Ingenuity pathways analysis (IPA) was used to identify involved pathways. The results show that GHB induces gene expression alterations in hundreds of genes in the hippocampus, cortex and striatum, and the number of affected genes increases throughout a 4-h time course. Many of these DEGs are involved in neurological disease, apoptosis, and oxidative stress.

Reconstitution of centrosome microtubule nucleation in Spisula.

Evidence strongly supports the hypothesis that gamma-TuRCs are sites for the nucleation of microtubules within the centrosome PCM (Zheng et al., 1995; Moritz et al., 1995a,b). Further, these structures appear to be recruited to the centrosome from cytoplasmic pools during centrosome assembly and centrosome maturation (Moritz et al., 1998; Schnackenberg et al., 1998, 2000; Schnackenberg and Palazzo, 1999; Khodjakov and Rieder, 1999). It has also been shown that the centrosome PCM contains a network of filamentous fibers, which function as a scaffold for binding these microtubule nucleating sites (Schnackenberg et al., 1998). However, binding gamma-tubulin rings to this scaffold requires at least one additional factor (Moritz et al., 1998; Schnackenberg et al., 2000). Because extracts prepared from a variety of biological sources are capable of supporting the recovery of microtubule nucleation by Spisula KICRs (Schnackenberg et al., 2000), the methods outlined in this chapter could prove useful in the search for factors that are necessary for centrosome assembly and the increase in centrosome-dependent microtubule nucleation during centrosome maturation.

Cyclin E and its associated cdk activity do not cycle during early embryogenesis of the sea Urchin.

Female sea urchins store their gametes as haploid eggs. The zygote enters S-phase 1 h after fertilization, initiating a series of cell cycles that lack gap phases. We have cloned cyclin E from the sea urchin Strongylocentrotus purpuratus. Cyclin E is synthesized during oogenesis, is present in the germinal vesicle, and is released into the egg cytoplasm at oocyte maturation. Cyclin E synthesis is activated at fertilization, although there is no increase in cyclin E protein levels due to continuous turnover of the protein. Cyclin E protein levels decline in morula embryos, while cyclin E mRNA levels remain high. After the blastula stage, cyclin E mRNA and protein levels are very low, and cyclin E expression is predominant only in cells that are actively dividing. These include cells in the left coelomic pouch, which forms the adult rudiment in the embryo. The cyclin E present in the egg is complexed with a protein kinase. Activity of the cyclin E/cdk2 changes little during the initial cell cycles. In particular, cyclin E-cdk2 levels remain high during both S-phase and mitosis. Our results suggest that progression through the early embryonic cell cycles in the sea urchin does not require fluctuations in cyclin E kinase activity.

Centrosome maturation.

In the past, centrosome maturation has been described as the change in microtubule nucleation potential that occurs as cells pass through specific phases of the cell cycle. It is suggested that the idea of centrosome maturation be expanded to include gain of functions that are not necessarily related to microtubule nucleation. Some of these functions could be transient and dependent on the temporary association of molecules with the centrosome as cells progress through the cell cycle. Thus, the centrosome may best be viewed as a site for mediating macromolecular interactions, perhaps as a central processing station within the cell. The centromatrix, a relatively stable lattice of polymers within the centrosome's PCM, could serve as a scaffold for the transient binding of mediator molecules, as well as allow the dynamic exchange of centrosome constituents with a soluble cytoplasmic pool. New evidence adds support to the idea that centrioles are crucial for the maintenance of PCM structure. However, significant evidence indicates that aspects of centrosome structure and function can be maintained in the absence of centrioles. In the case of paternal centrosome maturation, sperm centrioles may not contain an associated centromatrix. It is proposed that regulation of paternal centrioles or centriole associated proteins could mediate centriole-dependent centromatrix assembly following fertilization. Thus, regulation of centromatrix-centriole interactions could be involved in maintaining the integrity of the centrosome's PCM and play an important role in centrosome disassembly during cell differentiation and morphogenesis.

Reconstitution of microtubule nucleation potential in centrosomes isolated from Spisula solidissima oocytes.

Treatment of isolated Spisula solidissima centrosomes with KI removes (gamma)-tubulin, 25 nm rings, and their microtubule nucleation potential, revealing the presence of a filamentous lattice, the 'centromatrix'. Treatment of this centromatrix with Spisula oocyte extract results in the binding of (gamma)-tubulin and 25 nm rings, and the recovery of microtubule nucleation potential. Fractionation of this extract resulted in the separation of elements that are required for the recovery of microtubule nucleation potential. We show that some, but not all, of the elements needed cosediment with microtubules. Further, extracts prepared from activated (meiotic) and non-activated (interphase) Spisula oocytes, CHO cells blocked in S phase, Drosophila embryos and Xenopus oocytes all support the recovery of microtubule nucleation potential by the Spisula centromatrix. These results demonstrate that components necessary for centrosome-dependent microtubule nucleation are functionally conserved and abundant in both interphase and meiotic/mitotic cytoplasm.

Identification and function of the centrosome centromatrix.

Centrosomes direct the organization of microtubules in animal cells. However, in the absence of centrosomes, cytoplasm has the potential to organize microtubules and assemble complex structures such as anastral spindles. During cell replication or following fertilization, centrioles that are incapable of organizing microtubules into astral arrays are introduced into this complex cytoplasmic environment. These centrioles become associated with pericentriolar material responsible for centrosome-dependent microtubule nucleation, and thus the centrosome matures to ultimately become a dominant microtubule organizing center that serves as a central organizer of cell cytoplasm. We describe the identification of a novel structure within the pericentriolar material of centrosomes called the centromatrix. The centromatrix is a salt-insoluble filamentous scaffold to which subunit structures that are necessary for microtubule nucleation and abundant in the cytoplasm bind. We propose that the centromatrix serves to concentrate and focus these subunits to form the microtubule organizing center. Since binding of these subunits to the centromatrix does not require nucleotides, we propose a model for centrosome assembly which predicts that the assembly of the centromatrix is a rate-limiting step in centrosome assembly and maturation.

The disassembly and reassembly of functional centrosomes in vitro.

Animal cells contain a single centrosome that nucleates and organizes a polarized array of microtubules which functions in many cellular processes. In most cells the centrosome is composed of two centrioles surrounded by an ill-defined "cloud" of pericentriolar material. Recently, gamma-tubulin-containing 25-nm diameter ring structures have been identified as likely microtubule nucleation sites within the pericentriolar material of isolated centrosomes. Here we demonstrate that when Spisula centrosomes are extracted with 1.0 M KI they lose their microtubule nucleation potential and appear by three-dimensional electron microscopy as a complex lattice, built from 12- to 15-nm thick elementary fiber(s), that lack centrioles and 25-nm rings. Importantly, when these remnants are incubated in extracts prepared from Spisula oocytes they recover their 25-nm rings, gamma-tubulin, and microtubule nucleation potential. This recovery process occurs in the absence of microtubules, divalent cations, and nucleotides. Thus, in animals the centrosome is structurally organized around a KI-insoluble filament-based "centromatrix" that serves as a scaffold to which those proteins required for microtubule nucleation bind, either directly or indirectly, in a divalent cation and nucleotide independent manner.