Assembly of vault-like particles in insect cells expressing only the major vault protein.

Vaults are the largest (13 megadalton) cytoplasmic ribonucleoprotein particles known to exist in eukaryotic cells. They have a unique barrel-shaped structure with 8-fold symmetry. Although the precise function of vaults is unknown, their wide distribution and highly conserved morphology in eukaryotes suggests that their function is essential and that their structure must be important for their function. The 100-kDa major vault protein (MVP) constitutes approximately 75% of the particle mass and is predicted to form the central barrel portion of the vault. To gain insight into the mechanisms for vault assembly, we have expressed rat MVP in the Sf9 insect cell line using a baculovirus vector. Our results show that the expression of the rat MVP alone can direct the formation of particles that have biochemical characteristics similar to endogenous rat vaults and display the distinct vault-like morphology when negatively stained and examined by electron microscopy. These particles are the first example of a single protein polymerizing into a non-spherically, non-cylindrically symmetrical structure. Understanding vault assembly will enable us to design agents that disrupt vault formation and hence aid in elucidating vault function in vivo.

Up-regulation of vaults may be necessary but not sufficient for multidrug resistance.

Vaults are ribonucleoprotein complexes comprised of the 100 kDa major vault protein (MVP), the 2 high m.w. vault proteins p193 (VPARP) and p240 (TEP1) and an untranslated small RNA (vRNA). Increased levels of MVP, vault-associated vRNA and vaults have been linked directly to non-P-glycoprotein-mediated multidrug resistance (MDR). To further characterize the putative role of vaults in MDR, expression levels of all of the vault proteins were examined in various MDR cell lines. Subcellular fractionation of vault particles revealed that all 3 vault proteins are increased in MDR cells compared to the parental, drug-sensitive cells. Furthermore, protein analysis of subcellular fractions of the drug-sensitive, MVP-transfected AC16 cancer cell line indicated that vault levels are increased, in this stable line. Since TEP1 is shared by both vaults and the telomerase complex, TEP1 protein (and vault) levels were compared with telomerase activity in a variety of cell lines, including various MDR lines. Our studies demonstrate that while vault levels may be a good predictor of drug resistance, their up-regulation alone is not sufficient to confer the drug-resistant phenotype. This implies a requirement of an additional factor(s) for vault-mediated MDR.

Analysis of sulfatide from rat cerebellum and multiple sclerosis white matter by negative ion electrospray mass spectrometry.

The accumulation of sulfatide (sulfatogalactosyl cerebroside) and changes in the sulfatide species present have been examined in the cerebellum of day 6-32 aged rats and in multiple sclerosis (MS) tissue samples. Negative ion electrospray mass spectrometry with daughter and parent ion analyses were used to distinguish the fatty acyl character in the amide linkage of sulfatide; measurement was done by selected ion and multiple reaction monitoring of individually identified sulfatide molecules. Sulfatide accumulation in rat cerebellum shows that 18:0- and hydroxylated 18:0-sulfatide are the first sulfatide molecules detectable. Very long fatty acyl chain sulfatide molecules (>20:0) are present at day 7 and the ratio of non-hydroxylated compared to hydroxylated sulfatide rises as the amount of non-hydroxylated sulfatide increases. 24:1-sulfatide accumulates at a ratio of about 3:1 over 24:0-sulfatide during active myelination. Analyses of the sulfatide in human tissue have shown differences between MS plaque tissues, normal appearing adjacent white matter and control tissues. The findings show that total sulfatide is reduced by 60% in the plaque matter and decreased 25% in adjacent normal appearing white matter. There are significant increases (P=0.05) in the amount of hydroxylation of sulfatide, demonstrated by an increase in the percentage of hydroxylated h24:0-sulfatide (hydroxy-lignoceroyl sulfatide).

Cloning of a cDNA encoding a sequence-specific single-stranded-DNA-binding protein from Rattus norvegicus.

In this paper, we report the isolation of a cDNA clone encoding a sequence-specific single-stranded-DNA-binding protein (SSDP) from rat (Rattus norvegicus). The full-length nucleotide sequence was determined and encodes a 361 amino acid protein with a predicted molecular mass of 37.7 kDa. This clone has approximately 80% homology to a previously isolated partial cDNA clone for SSDP from chicken (Gallus gallus). Northern blot analysis revealed two transcripts of 2.0 and 3.0 kb. The protein appears to be evolutionarily highly conserved with > 97% identity between chicken, rat, mouse and human. Chicken SSDP has been proposed to be involved in the transcriptional regulation of the alpha 2(I) collagen gene.

Role of axonal components during myelination.

Myelination is a multistep ordered process whereby Schwann cells in the peripheral nervous system (PNS) and oligodendrocytes in the central nervous system (CNS), produce and extend membranous processes that envelop axons. Mechanisms that regulate this complex process are not well understood. Advances in deciphering the regulatory components of myelination have been carried out primarily in the PNS and although the mechanisms for triggering and directing myelination are not known, it is well established that myelination does not occur in the absence of axons or axon/neuron-derived factors. This appears to be true both in PNS and CNS. Progress in understanding CNS myelinogenesis has been relatively slow because of the unavailability of a suitable culture system, which, in turn, is partly due to complexity in the cellular organization of the CNS. Though the myelin composition differs between PNS and CNS, the regulation of myelination seems to parallel rather than differ between these two systems. This article reviews the regulatory role of axonal components during myelination. The first half consists of an overview of in vitro and in vivo studies carried out in the nervous system. The second half discusses the use of a cerebellar slice culture system and generation of anti-axolemma monoclonal antibodies to investigate the role of axonal membrane components that participate in myelination. It also describes the characterization of an axonal protein involved in myelination.

Axonal proteins involved in myelination: characterization of a collagen-like protein.

An anti-axolemma monoclonal antibody, designated G21.3, has been isolated in order to understand molecular mechanisms involved in myelination. Both biochemical and morphological studies showed that the monoclonal antibody inhibits myelin production by oligodendrocytes in cerebellar slice cultures. On Western blots of axolemma preparations, the antibody recognized 140- and 120-kD proteins. The present study involves the isolation and characterization of the G21.3 antigen. The G21.3-immunoreactive proteins of 140 and 120 kD were purified from the adult rat sciatic nerve and amino acid sequencing of these proteins revealed significant homology to alpha I and alpha II chains of collagen type I. Biochemical and Western blot analysis using pure collagen, collagen I antibody and collagenase D suggest that the antigen isolated from sciatic nerve is collagen. However, immunofluorescence studies using the G21.3 antibody, collagen I antibody, collagenase D and Northern blot analysis using a collagen probe do not fully support the view that the G21.3 antigen in the CNS is also a collagen. We conclude that the G21.3 antigen is a collagen-like protein involved in CNS myelination.