Impact system for ultrafast synchrotron experiments.

The impact system for ultrafast synchrotron experiments, or IMPULSE, is a 12.6-mm bore light-gas gun (<1 km/s projectile velocity) designed specifically for performing dynamic compression experiments using the advanced imaging and X-ray diffraction methods available at synchrotron sources. The gun system, capable of reaching projectile velocities up to 1 km/s, was designed to be portable for quick insertion/removal in the experimental hutch at Sector 32 ID-B of the Advanced Photon Source (Argonne, IL) while allowing the target chamber to rotate for sample alignment with the beam. A key challenge in using the gun system to acquire dynamic data on the nanosecond time scale was synchronization (or bracketing) of the impact event with the incident X-ray pulses (80 ps width). A description of the basic gun system used in previous work is provided along with details of an improved launch initiation system designed to significantly reduce the total system time from launch initiation to impact. Experiments were performed to directly measure the gun system time and to determine the gun performance curve for projectile velocities ranging from 0.3 to 0.9 km/s. All results show an average system time of 21.6 ± 4.5 ms, making it possible to better synchronize the gun system and detectors to the X-ray beam.

Expression and purification of the extracellular domain of human myelin protein zero.

Myelin protein zero (P0), an adhesion protein of the immunoglobulin superfamily, is the major protein of peripheral nervous system myelin in higher vertebrates. Protein zero is required for the formation and maintenance of myelin structure in the internode, likely through homophilic interactions at both the extracellular and the intracellular domains. Mutations and deletions in the P0 gene correlate with hereditary peripheral neuropathies of varying severity. Comparisons between the human and rat isoforms, whose three-dimensional structure has been determined by X-ray crystallography, suggest that these disease-associated genetic alterations lead to structural changes in the protein that alter P0-P0 interactions and hence affect myelin functionality. Knowing the crystal structures of native and altered human P0 isoforms could help to elucidate the structural changes in myelin membrane packing that underlie the altered functionality. Alterations of P0 extracellular domain (P0-ED) are of additional interest as previous X-ray diffraction studies on myelin membrane packing suggest that P0-ED molecules can assume distinct adhesive arrangements. Here, we describe an improved method to express and purify human P0-ED (hP0-ED) suitable for crystallographic analysis. A fusion protein consisting of maltose binding protein fused to hP0-ED was secreted to the periplasm of Escherichia coli to allow an appropriate folding pathway. The fusion protein was extracted via osmotic shock and purified by affinity chromatography. Factor Xa was used to cleave the fusion protein, and a combination of affinity and ion-exchange chromatography was used to further purify hP0-ED. We document several significant improvements to previous protocols, including bacterial growth to approximately 15 OD using orbital shakers and the use of diafiltration, which result in yields of approximately 150 mg highly pure protein per liter of medium.

Single injections of a DNA plasmid that contains the human Bcl-2 gene prevent loss and atrophy of distinct neuronal populations after spinal cord injury in adult rats.

Spinal cord injury in adult mammals causes atrophy or loss of axotomized neurons. We have previously found that the product of the antiapoptotic gene Bcl-2, delivered by intraspinal injection of a DNA plasmid, reduces atrophy and loss of axotomized Clarke's nucleus neurons in adult rats. Here we studied whether the same treatment protects axotomized red nucleus (RN) neurons. Two months after the right dorsolateral funiculus was ablated in adult Sprague-Dawley rats by C3/C4 subtotal hemisection, there was approximately 48% loss of RN neurons in the magnocellular portion of the RN contralateral to the lesion and atrophy of many surviving neurons. When a DNA plasmid encoding the human Bcl-2 gene and the bacterial reporter gene LacZ, complexed with cationic lipids, was injected just rostral to the subtotal hemisection site, 87% of RN neurons survived, and there was partial, but robust, protection from atrophy. These and our previous results indicated that intraspinal administration of the Bcl-2 gene can prevent retrograde cell loss and reduce atrophy of axotomized RN and Clarke's nucleus neurons in adult rats and provide an effective means to rescue neurons whose survival depends on different growth factors.

Red nucleus neurons of Bcl-2 over-expressing mice are protected from cell death induced by axotomy.

The Bcl-2 proto-oncogene regulates apoptosis and prevents cell death. We studied the effect of Bcl-2 gene over-expression on the survival of axotomized red nucleus (RN) neurons after unilateral hemisection at cervical segment 4/5 (C4/5) in mice. Seventy-five percent of RN neurons survived in Bcl-2 over-expressing mice 1 or 2 months after surgery compared with only 55% of RN neurons in wild-type mice. However, Bcl-2 gene over-expression does not prevent lesion-induced shrinkage of RN neurons.

DNA plasmid that codes for human Bcl-2 gene preserves axotomized Clarke's nucleus neurons and reduces atrophy after spinal cord hemisection in adult rats.

Spinal cord injury in adult mammals causes atrophy or death of some axotomized neurons. The product of the antiapoptotic gene Bcl-2 prevents neuron death in vivo. We delivered Bcl-2 by intraspinal injection of a DNA plasmid encoding this gene to determine if axotomized neurons destined to undergo retrograde death could be rescued. Axons of the right side Clarke's Nucleus (CN) were cut unilaterally in adult Sprague-Dawley rats by T8 hemisection, leaving the contralateral (left) CN as an intact control. Two months postoperatively, there was approximately 35% loss of total CN neurons in the right L1 segment. Only 15% of large CN neurons (>400 microm2), whose axons project to the cerebellum, survived--indicating atrophy and/or death of 85% of these cells. We injected a DNA plasmid encoding the human Bcl-2 gene and the bacterial reporter gene LacZ, which was complexed with cationic lipids, into the right side of segment T8 of the normal spinal cord, or just caudal to the hemisection site. The reporter gene was expressed in the perikarya of right CN neurons at L1 for up to 7 days, but not 14 days. Two months following T8 hemisection and Bcl-2/LacZ DNA injection, there was no significant loss of CN neurons ipsilateral to the lesion. Surprisingly, 61% of large neurons survived, indicating partial protection from atrophy. In contrast, a DNA plasmid that codes for the LacZ reporter gene, but not Bcl-2, did not prevent CN neuron death or atrophy. Administration of the Bcl-2 gene in adult rats and its expression in these CNS neurons prevents retrograde cell death, and also minimizes atrophy. These results may serve as the basis for developing novel gene therapy strategies for patients with spinal cord injury.

Two proteins bind to a novel motif in the promoter of the myelin basic protein gene from mouse.

The box 1 and 2 motif of the myelin basic protein (MBP) promoter is a potential regulatory sequence of the MBP transcription unit. A DNA fragment that contained the sequence of the box 1 and 2 motif from mouse was synthesized, and its protein binding properties were examined by gel-shift assays. The box 1 and 2 probe and nuclear extracts from mouse brain generated a pattern of six major DNA-protein complexes (a, b, c, d, e, and f). The box 1 and 2 probe and nuclear extracts from oligodendrocyte-like glioma cells 1C10 generated a pattern of DNA-protein complexes that exhibited only complexes a, b, e, and f. Complex b generated by extracts from 1C10 cells, however, was very intense compared to any of the other complexes. It was determined that dephosphorylation of the proteins in nuclear extracts from 1C10 cells with acid phosphatase significantly altered their DNA binding properties. Two proteins of minimum M, approximately 32 and approximately 38 kDa (MBP32 and MBP38) that bind to the box 1 and 2 motif were identified in these nuclear extracts by using a UV crosslinking method. MBP32 and MBP38 are found in cell types and tissues known to express the golli transcription unit of the golli-MBP gene complex and may be involved in the modulation of the MBP unit in those cells.

Membrane adhesion in peripheral myelin: good and bad wraps with protein P0.

Recent molecular models and crystallographic analysis of the major protein of peripheral myelin have provided new insights into the molecular basis of membrane adhesion in myelin. These studies have proved useful in understanding the molecular basis of clinical phenotypes in certain demyelinating neuropathies.

Topological complexity of SV40 minichromosomes.

During attempts to measure the extent to which the proteins of simian virus 40 (SV40) minichromosomes restrain the ability of SV40 DNA to alter its twist in response to temperature changes, we found that temperature-shift-induced linking number changes are not reversible for isolated minichromosomes, suggesting that such changes, both in isolated minichromosomes and in cells, may be a consequence of structural alterations in chromatin proteins rather than of simple changes in DNA twist. We also found that the SV40 minichromosome pool is composed of subpopulations that display different responses to temperature shifts. For example, the linking number of DNA in newly replicated minichromosomes is more responsive to in vivo temperature changes than is the linking number of DNA in bulk minichromosomes. In addition, the linking number profiles of both isolated and intracellular minichromosomes change during the course of infection. These observations emphasize the topological complexity of SV40 minichromosomes and encourage caution in the interpretation of experiments carried out on bulk minichromosomes.

Gene expression and phosphorylation of mouse osteopontin.

Osteopontin is expressed in many different cell types and has been proposed to play several functions. Distinct forms of the protein have been detected. Various tissues and cell lines from mouse, however, exhibit two classes of transcripts with different 5'-untranslated ends but with an identical coding region (exons II through VII). These transcripts do not arise from the alternative splicing of coding exons. These results suggest that posttranslational modifications of osteopontin, such as phosphorylation, are a major mechanism to generate different forms of the protein. Mouse osteopontin was expressed in E. coli and used as a model to study its phosphorylation.

The roles of autophosphorylation and phosphorylation in the life of osteopontin.

Osteopontin is a secreted glycosylated phosphoprotein found in bone and other normal and malignant tissues. Osteopontin can be autophosphorylated on tyrosine residues and can also be phosphorylated on serine and threonine residues by several protein kinases. Autophosphorylation of osteopontin may generate sites for specific interactions with other proteins on the cell surface and/or within the extracellular matrix. These interactions of osteopontin are thought to be essential for bone mineralization and function. The polyaspartic acid motif of osteopontin, in combination with neighboring sequences that include serine residues phosphorylated by protein kinases, could fold and assemble into a molecular structure that participates in the mineralization of the bone matrix.

Mutations in demyelinating peripheral neuropathies support molecular model of myelin P0-glycoprotein extracellular domain.

Homophilic interactions of the major integral membrane protein of peripheral nerve myelin, P0-glycoprotein, are thought to mediate membrane adhesion and compaction. Molecular modeling of its extracellular domain (P0-ED), based on its resemblance to an immunoglobulin variable domain and on X-ray diffraction measurements of inter-membrane spacings of myelin, has suggested which amino acid sidechains may be involved in the homophilic adhesion. Recently identified point-mutations in the human P0 gene result in amino acid substitutions in P0 protein and correlate with demyelinating motor and sensory neuropathies. The molecular model explains how these changes result in disrupted P0-P0 interactions; indicates how compensatory changes in amino acids, as occur in P0-ED of other species, preserve normal homophilic interactions; and predicts what other residue substitutions might underlie additional cases of demyelinating neuropathies.

Box I and II motif from myelin basic protein gene promoter binds to nuclear proteins from rodent brain.

The box I and II motif located within the promoter of the myelin basic protein gene contains a simian virus 40 T-antigen-binding site, a MyoD/E2a-binding site, and a glucocorticoid receptor-binding site. We have found proteins within nuclear extracts from adult mouse brain, rat embryonic cerebral cortex in culture, and a mouse oligodendrocyte-like cell line that bind to a 32P-labeled synthetic DNA fragment containing the sequences of the box I and II motif. Three major complexes (A, B, and C) were seen in gel-shift assays. Only complexes A and B were competed out by the unlabeled box I and II fragment or by another synthetic DNA fragment that also contains sequences similar to a glucocorticoid receptor-binding site. Therefore, complexes A and B were thought to be specific. The expression pattern of the proteins responsible for the formation of these complexes was also assessed during development in mouse brain.

Myelin P0-glycoprotein: predicted structure and interactions of extracellular domain.

Protein zero (P0), a transmembrane glycoprotein, accounts for over 50% of the total protein in PNS myelin. The extracellular domain of P0 (P0-ED) is similar to the immunoglobulin variable domain, carrying one acceptor sequence for N-linked glycosylation. The x-ray diffraction analysis of PNS myelin has demonstrated reversible transitions that depend on pH and ionic strength, resulting in three distinct structures characterized by widths of about 36 A, 50 A (native), and 90 A between the extracellular surfaces of the membranes. In the current work, we considered the constraints imposed by these x-ray diffraction data on the orientation of P0-ED, and we propose how this immunoglobulin-like domain could be accommodated in the variable widths of the extracellular space between myelin membranes. The modeling made use of the finding that beta-strand predictions for P0-ED are virtually superimposable with those of the VH domain of the phosphocholine-binding immunoglobulin M603 of mouse, which has a similar number of residues as P0-ED and a structure that has been solved crystallographically. The dimensions of P0-ED from the space-filling model, developed using PC-based molecular modeling software, were found to be 44 A x 25 A x 23 A. On the assumption that neither the shape nor the orientation of P0-ED changes appreciably, then the different widths at the extracellular apposition would easily accommodate P0-ED from apposed membranes if the molecules were oriented so that the beta-strands were approximately perpendicular to the membrane surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning of the myelin basic proteins in the shark, Squalus acanthias, and the ray, Raja erinacia.

Myelin basic proteins (MBPs) are a family of alternatively spliced isoforms present in myelin sheaths of most vertebrates. A reverse transcriptase-polymerase chain reaction (RT-PCR) approach was used to clone MBP isoforms in species representing two superorders of elasmobranchs: Squalus acanthias, representing Squalomorph sharks, and Raja erinacia, representing Batoidea rays. Two products were generated from each species. The larger product encoded a 155 amino acid protein, the same size as MBPs from two Galeomorph sharks, Heterodontus francisci and Carcharhinus obscurus, which, based upon alignment with other vertebrate MBPs, contained six of the seven MBP exons; only exon II was absent. The smaller product encoded a 141 amino acid protein that lacked exon II and exon V. There were 26 and 30 nucleotide differences between Squalus and Heterodontus, and Raja and Heterodontus, respectively. Sequences from Squalus and Raja were far more similar, having only five nucleotide differences. Both isoforms of elasmobranch MBP contain 18.5% basic (lysine plus arginine) amino acids, compared with 17.5% in mammalian MBPs comprised of the corresponding exons. Northern blot analysis of whole brain total RNA revealed a single band of 2.5 kb in Squalus, and three bands of 1.2, 1.4, and 2.3 kb in Raja. The finding that MBPs of a Squalomorph shark and a Batoidea ray are closer to one another than either is to the Galeomorph sharks suggests that MBP sequence information may prove useful in classifying modern day Chondrichthytes.

Mouse osteopontin expressed in E. coli exhibits autophosphorylating activity of tyrosine residues.

Osteopontin is a secreted glycosylated phosphoprotein found in various normal and transformed tissues. Mouse osteopontin expressed in bacteria has been found to autophosphorylate in vitro using ATP or GTP as phosphoryl donors. The reaction does not occur using inorganic orthophosphate as the donor. Only tyrosine residues are phosphorylated. Neither serine nor threonine residues, both of which are found phosphorylated in osteopontin extracted from bone, is autophosphorylated in vitro. The autophosphorylation of tyrosine residues by a secreted protein such as osteopontin may provide additional insight into its biological functions.

In vitro phosphorylation of mouse osteopontin expressed in E. coli.

To understand the role of post-translational modifications on the structure and function of osteopontin, a secreted glycosylated phosphoprotein, we expressed mouse osteopontin in E. coli as a fusion protein with glutathione-S-transferase (GST). The purified fusion protein was cleaved by factor Xa generating GST (26 kDa) and recombinant osteopontin (60 kDa). The fusion protein was phosphorylated in vitro by cytosolic, microsomal, and casein kinase II fractions from mouse kidney homogenates. The fusion protein and recombinant osteopontin were also phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. The suitability of the fusion and recombinant proteins as model substrates for the study of the function(s) and post-translational modifications of osteopontin is discussed.