Lysenin is a novel protein derived from coelomic fluid of the earthworm Eisenia foetida, which specifically recognizes sphingomyelin and induces cytolysis. The mechanism underlying lysenin-induced cell lysis has not been clarified. In this report we studied the interaction of lysenin with red blood cells as well as artificial liposomes. Our results showed that lysenin bound membranes and assembled to SDS-resistant oligomers in a sphingomyelin-dependent manner, leading to the formation of pores with a hydrodynamic diameter of ϳ3 nm. Antibody scanning analysis suggested that the Cterminal region of lysenin was exposed, whereas the N-terminal was hidden in the isolated oligomer complex. Differential scanning calorimetry revealed that lysenin interacted with both hydrophilic head group and hydrophobic hydrocarbon tails of sphingomyelin. Oligomerization but not binding was affected by the amide-linked fatty acid composition of sphingomyelin, suggesting the role of membrane fluidity in the oligomerization step.
DNA-negative Dane particles have been observed in hepatitis B virus (HBV)-infected sera.The capsids of the empty particles are thought to be composed of core protein but have not been studied in detail. In the present study, the protein composition of the particles was examined using new enzyme immunoassays for the HBV core antigen (HBcAg) and for the HBV precore/core proteins (core-related antigens, HBcrAg). HBcrAg were abundant in fractions slightly less dense than HBcAg and HBV DNA. Three times more Dane-like particles were observed in the HBcrAg-rich fraction than in the HBV DNA-rich fraction by electron microscopy. Western blots and mass spectrometry identified the HBcrAg as a 22-kDa precore protein (p22cr) containing the uncleaved signal peptide and lacking the arginine-rich domain that is involved in binding the RNA pregenome or the DNA genome. In sera from 30 HBV-infected patients, HBcAg represented only a median 10.5% of the precore/ core proteins in enveloped particles. These data suggest that most of the Dane particles lack viral DNA and core capsid but contain p22cr. This study provides a model for the formation of the DNA-negative Dane particles. The precore proteins, which lack the arginine-rich nucleotide-binding domain, form viral RNA/DNA-negative capsid-like particles and are enveloped and released as empty particles. Hepatitis B virus (HBV)1 infects more than 300 million people and is a major cause of liver diseases. The HBV belongs to the Hepadnavirus family and is a small (42 nm) enveloped DNA virus, which possesses a 27-nm icosahedral nucleocapsid composed of core protein and a 3.2-kb partially doublestranded, circular genome (1). Although the term "Dane particles" refers to the 42-nm HBV particles (2) and is often used in reference to the complete HBV particles, electron microscopic studies have suggested that the DNA-negative "empty" Dane particles are predominant in sera (3-6). The capsids of the empty particles are thought to be composed of core protein but have not been studied in detail.The HBV genome encodes two core-related open reading frames, precore and core genes (Fig. 1). These are expressed because of two in-frame ATG initiation codons located at the 5Ј end of the genes. The first ATG encodes a 25-kDa protein (p25) containing the 29-amino acid (aa) precore sequence fused to the N terminus of the HBV core antigen (HBcAg). The p25 is directed toward the secretory pathway by a 19-aa signal sequence that is cleaved during translocation into the lumen of the endoplasmic reticulum (ER), producing a 22-kDa protein. Subsequent proteolytic cleavages within the arginine-rich Cterminal region (34 aa) generate a 17-kDa protein that is secreted as hepatitis B e antigen (HBeAg) (7-10). A heterogeneous population of these precore derivatives has been observed in the sera of patients and is serologically defined as HBeAg (9,11,12). Conversely, the second ATG specifies the 21.5-kDa HBcAg, which assembles into dimers that form the virus capsid (7,9,(13)(14)(15). HBcAg is a 183-residue protein wi...
Morphological studies using immersion or perfusion fixation methods do not reveal the ultrastructure of functioning kidneys with normal circulation. A simple apparatus was developed for freezing the kidneys in vivo without stopping the blood supply, and the ultrastructure of the glomerular capillary loops was examined under different haemodynamic conditions. Mouse kidneys were frozen under normal blood flow conditions; others were frozen in the same way after ligation of the abdominal aorta at a point caudal to the renal arteries. They were then processed for the freeze-substitution or deep-etching method. Good ultrastructural preservation was obtained within about 5 microM depth from the frozen tissue surface. Functioning glomeruli with normal blood flow possessed open capillary lumens, different shapes of foot processes and atypical basement membranes with low density. Moreover, heterogeneity in width between foot processes was identified on the replica membranes. Under the acute conditions used to increase blood supply into the kidneys, the spaces between the flat foot processes became more widely dilated and the basement membrane was seen to be three-layered. The ultrastructure of glomeruli in functioning kidneys has been demonstrated for the first time by this "in vivo cryotechnique."
Axonal pathology is a major cause of neurological disability in multiple sclerosis. Axonal transection begins at disease onset but remains clinically silent because of compensatory brain mechanisms. Noninvasive surrogate markers for axonal injury are therefore essential to monitor cumulative disease burden in vivo. The neuronal compound N-acetylaspartate, as measured by magnetic resonance spectroscopy, is currently the best and most specific noninvasive marker of axonal pathology in multiple sclerosis. The possibility has been raised, however, that N-acetylaspartate is expressed also by oligodendroglial lineage cells. In order to investigate N-acetylaspartate specificity for white matter axons, transected rat optic nerves were analyzed by high-performance liquid chromatography and immunohistochemistry. In transected adult nerves, N-acetylaspartate and N-acetyl aspartylglutamate decreased in concordance with axonal degeneration and were undetectable 24 days posttransection. Nonproliferating oligodendrocyte progenitor cells, oligodendrocytes, and myelin were abundant in these axon-free nerves. At 24 days posttransection, N-acetylaspartate was increased (42%; p = 0.02) in nontransected contralateral nerves. After transection at postnatal day 4, total N-acetylaspartate decreased by 80% (P14; p = 0.002) and 94% (P20; p = 0.003). In these developing axon-free nerves, 25 to 33% of oligodendrocyte progenitor cells were proliferating. These data validate magnetic resonance spectroscopy measurements of N-acetylaspartate as an axon-specific monitor of central nervous system white matter in vivo. In addition, the results indicate that neuronal adaptation can increase N-acetylaspartate levels, and that 5 to 20% of the N-acetylaspartate in developing white matter is synthesized by proliferating oligodendrocyte progenitor cells.
Previous studies identified the tetraspanin protein CD9 in myelinating oligodendrocytes. The present report extends these observations by identifying CD9 in a subpopulation of oligodendrocyte progenitor cells (OPCs) and in premyelinating oligodendrocytes in rodents. NG2-positive cells expressed CD9 in a temporal and spatial pattern during development that was consistent with CD9 expression in OPCs just prior to their differentiation into premyelinating oligodendrocytes. NG2-positive cells in mature brains were CD9-negative. CD9 expression during oligodendrocyte development in vitro supported this hypothesis, as all CD9-positive cells became O4-positive when switched to oligodendrocyte differentiating media. CD9 immunoreactivity was enriched in myelinating oligodendrocytes and their processes, and the outer aspects of myelin internodes. Immunoprecipitation of CD9 from postnatal rat cerebrum coprecipitated beta1 integrin, CD81, and Tspan-2, another tetraspanin protein recently identified in oligodendrocytes. Following surface biotinylation of oligodendrocytes in vitro, biotinylated beta1 integrin was identified in a CD9 immunoprecipitate. These data support a molecular link between surface integrins and a CD9, Tspan-2 molecular web during the differentiation of oligodendrocytes. Oligodendrocyte production and myelination appears to be normal in CD9-deficient mice. These data support the hypothesis that CD9 helps form the tetraspanin web beneath the plasma membranes of progenitor cells committed to oligodendrogenesis, but that CD9 is not essential for oligodendrogenesis and myelination.
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