Biological and biochemical characteristics of prion strains conserved in persistently infected cell cultures

Abnormal prion protein (PrP(Sc)) plays a central role in the transmission of prion diseases, but the molecular basis of prion strains with distinct biological characteristics remains to be elucidated. We analyzed the characteristics of prion disease by using mice inoculated with the Chandler and Fukuoka-1 strains propagated in a cultured mouse neuronal cell line, GT1-7, which is highly permissive to replication of the infectious agents. Strain-specific biological characteristics, including clinical manifestations, incubation period as related to the infectious unit, and pathological profiles, remained unchanged after passages in the cell cultures. We noted some differences in the biochemical aspects of PrP(Sc) between brain tissues and GT1-7 cells which were unlikely to affect the strain phenotypes. On the other hand, the proteinase K-resistant PrP core fragments derived from Fukuoka-1-infected tissues and cells were slightly larger than those from Chandler-infected versions. Moreover, Fukuoka-1 infection, but not Chandler infection, gave an extra fragment with a low molecular weight, approximately 13 kDa, in both brain tissues and GT1-7 cells. This cell culture model persistently infected with different strains will provide a new insight into the understanding of the molecular basis of prion diversity.     

Hematopoietic tissue-specific expression of mouse Neil3 for endonuclease VIII-like protein

We cloned cDNA and genomic DNA containing exon 1 of mouse Neil3. Neil3 spans 52.4 kb and consists of 10 exons. Northern blot analysis revealed that Neil3 mRNA was selectively expressed in thymus, spleen and bone marrow. High levels of Neil3 mRNA were also detected in various mouse B cell lines by RT-PCR. Immunofluorescence microscopy using anti-NEIL3 revealed that recombinant mouse NEIL3 is localized in the nuclei. In mouse splenocytes, the level of Neil3 mRNA significantly increased after mitogen stimulation in vitro. We established NEIL3-null mice, which are viable and fertile. We found candidate sequences for NEIL3 orthologues in a DNA database from dog and zebrafish in addition to human and mouse, but not invertebrates. NEIL3 may function exclusively in vertebrates, such as mammals.     

Proteome analysis of human metaphase chromosomes

DNA is packaged as chromatin in the interphase nucleus. During mitosis, chromatin fibers are highly condensed to form metaphase chromosomes, which ensure equal segregation of replicated chromosomal DNA into the daughter cells. Despite >1 century of research on metaphase chromosomes, information regarding the higher order structure of metaphase chromosomes is limited, and it is still not clear which proteins are involved in further folding of the chromatin fiber into metaphase chromosomes. To obtain a global view of the chromosomal proteins, we performed proteome analyses on three types of isolated human metaphase chromosomes. We first show the results from comparative proteome analyses of two types of isolated human metaphase chromosomes that have been frequently used in biochemical and morphological analyses. 209 proteins were quantitatively identified and classified into six groups on the basis of their known interphase localization. Furthermore, a list of 107 proteins was obtained from the proteome analyses of highly purified metaphase chromosomes, the majority of which are essential for chromosome structure and function. Based on the information obtained on these proteins and on their localizations during mitosis as assessed by immunostaining, we present a four-layer model of metaphase chromosomes. According to this model, the chromosomal proteins have been newly classified into each of four groups: chromosome coating proteins, chromosome peripheral proteins, chromosome structural proteins, and chromosome fibrous proteins. This analysis represents the first compositional view of human metaphase chromosomes and provides a protein framework for future research on this topic.     

Bacillolysin MA, a novel bacterial metalloproteinase that produces angiostatin-like fragments from plasminogen and activates protease zymogens in the coagulation and fibrinolysis systems

We isolated a novel protease that converts plasminogen to angiostatin-like fragments (BL-angiostatins) from a culture of Bacillus megaterium A9542 through a single-step chromatography on CM-cellulose. The protease, designated bacillolysin MA (BL-MA), belongs to a family of neutral metalloproteinases based on the nucleotide sequence of its gene. At an enzyme:substrate ratio of 1:540, BL-MA cleaved human plasminogen mainly at Ser441-Val442 to form BL-angiostatin and miniplasminogen with a K(m) of 3.0 +/- 0.8 microM and a k(cat) of 0.70 +/- 0.09 s(-1). The resulting BL-angiostatins inhibited the proliferation, migration, and tube formation of vascular endothelial cells at concentrations of 1-10 microg/ml. Although BL-MA failed to activate plasminogen, it increased urokinase-catalyzed activation of plasminogen caused by production of miniplasminogen, which is highly susceptible to activation. In addition, BL-MA was active in converting prourokinase, prothrombin, coagulation factor X, and protein C to their active forms. BL-MA enhanced both the clotting of human plasma and clot dissolution in the presence of prourokinase. Thus, BL-MA affects blood coagulation and fibrinolysis systems and can be used to produce angiostatin-like plasminogen fragments and active serine proteases of human plasma.     

Localization of soluble beta-carbonic anhydrase in the marine diatom Phaeodactylum tricornutum. Sorting to the chloroplast and cluster formation on the girdle lamellae

A β-carbonic anhydrase (CA) in the marine diatom Phaeodactylum tricornutum (PtCA1) is encoded by the nuclear genome. This enzyme was previously found to be important for the operation of photosynthesis with a high affinity for dissolved inorganic carbon. A cDNA sequence that encodes PtCA1 (ptca1) was shown to possess a presequence of 138 bp (pre138), which encodes an N-terminal sequence of 46 amino acids (Pre46AA) that does not exist in the mature PtCA1. In this study, pre138 was ligated with the enhanced green fluorescent protein (GFP) gene (egfp), and introduced into P. tricornutum by microprojectile bombardment. Subsequently, the expressed Pre46AA-GFP fusion was shown to be localized in the chloroplast stroma, whereas the expressed GFP without Pre46AA was localized in the cytoplasm. Insertion of the DNA sequence, encoding a mature region of ptca1 (mptca1) between pre138 and egfp, resulted in the formation of particles with concentrated GFP fluorescence in the stroma of P. tricornutum. These particles, 0.3 to 3.0 μm in size, were shown to be distinct from the mitochondria and localized on the surface of the putative girdle lamella. The attachment of the initial one-half of the pre138 to the mptca1-egfp fusion caused the expressed GFP fusion to accumulate in areas surrounding the chloroplast, presumably due to the presence of the endoplasmic reticulum signal encoded by the initial half-sequence and to the absence of the chloroplast transit sequence. These results indicate that PtCA1 is targeted to the stroma by the bipartite sequences of Pre46AA and that the observed GFP particles are formed specifically in the stroma due to the function of the mptca1.     

Identification of a novel conjugate in human urine: bile acid acyl galactosides

We report a novel conjugate, bile acid acyl galactosides, which exist in the urine of healthy volunteers. To identify the two unknown peaks obtained in urine specimens from healthy subjects, the specimens were subjected to solid phase extraction and then to liquid chromatographic separation. The eluate corresponding to the unknown peaks on the chromatogram was collected. Following alkaline hydrolysis and liquid chromatography (LC)/electrospray ionization (ESI)-mass spectrometric (MS) analysis, cholic acid (CA) and deoxycholic acid (DCA) were identified as liberated bile acids. When a portion of the alkaline hydrolyzate was subjected to a derivatization reaction with 1-phenyl-3-methyl-5-pyrazolone, a derivative of galactose was detected by LC/ESI-MS. Finally, the liquid chromatographic and mass spectrometric properties of these unknown compounds in urine specimens were compared to those of authentic specimens and the structures were confirmed as CA 24-galactoside and DCA 24-galactoside. These results strongly imply that bile acid 24-galactosides, a novel conjugate, were synthesized in the human body.     

Tight junctions in Schwann cells of peripheral myelinated axons: a lesson from claudin-19-deficient mice

Tight junction (TJ)-like structures have been reported in Schwann cells, but their molecular composition and physiological function remain elusive. We found that claudin-19, a novel member of the claudin family (TJ adhesion molecules in epithelia), constituted these structures. Claudin-19-deficient mice were generated, and they exhibited behavioral abnormalities that could be attributed to peripheral nervous system deficits. Electrophysiological analyses showed that the claudin-19 deficiency affected the nerve conduction of peripheral myelinated fibers. Interestingly, the overall morphology of Schwann cells lacking claudin-19 expression appeared to be normal not only in the internodal region but also at the node of Ranvier, except that TJs completely disappeared, at least from the outer/inner mesaxons. These findings have indicated that, similar to epithelial cells, Schwann cells also bear claudin-based TJs, and they have also suggested that these TJs are not involved in the polarized morphogenesis but are involved in the electrophysiological "sealing" function of Schwann cells.     

Thioredoxin affinity chromatography: a useful method for further understanding the thioredoxin network

Thioredoxin affinity chromatography can be used to recognize the target proteins of thioredoxin or thioredoxin-related proteins in whole cells or certain cellular compartments. In the last couple of years, many potential target proteins have been identified from various organelles and organisms by this method. Based on the information on the target proteins provided by these studies, the complete thioredoxin-related redox networks can now be efficiently described.     

Genome-wide comparison of the His-to-Asp phosphorelay signaling components of three symbiotic genera of Rhizobia.

Histidine-to-aspartate (His-Asp) phosphorelay (or two-component) systems are very common signal transduction mechanisms that are implicated in a wide variety of cellular responses to environmental stimuli. The His-Asp phosphorelay components include "sensor histidine kinase (HK)", "phosphotransfer intermediate (HPt)", and "response regulator (RR)". With special reference to three bacterial species (Mesorhizobium loti, Bradyrhizobium japonicum, Sinorhizobium meliloti), each of which belongs to a different genera of Rhizobia, here we attempted to compile all of the His-Asp phosphorelay components in order to reveal a comparative genome-wide overview as to the His-Asp phosphorelay. It was revealed that M. loti has 47 HKs, 1 HPts, and 58 RRs; B. japonicum has 80 HKs, 3 HPts, and 91 RRs; whereas S. meliloti has 40 HKs, 1 HPt, and 58 RRs. These His-Asp phosphorelay components were extensively compiled and characterized. The resulting overview as to the His-Asp phosphorelay of Rhizobia will provide us with a basis for understanding of the fundamental mechanisms underlying interactions between plants and microorganisms (including symbiosis), as well as nitrogen fixation.     

Polyion complex micelles of pDNA with acetal-poly(ethylene glycol)-poly(2-(dimethylamino)ethyl methacrylate) block copolymer as the gene carrier system: physicochemical properties of micelles relevant to gene transfection efficacy

An acetal-poly(ethylene glycol)-poly(2-(dimethylamino)ethyl methacrylate) (acetal-PEG-PAMA) block copolymer spontaneously associated with plasmid DNA (pDNA) to form water-soluble complexes (polyion complex micelle: PIC micelle) in aqueous solution. Physicochemical characteristics and transfection efficiency of the PIC micelles thus prepared were studied here, focusing on the residual molar mixing ratio (N/P ratio) of AMA units in acetal-PEG-PAMA to the phosphate units in pDNA. With the N/P ratio increasing to unity, acetal-PEG-PAMA cooperatively formed complex micelles with pDNA through electrostatic interaction, allowing pDNA to condense effectively. Dynamic light scattering measurements revealed that the PIC micelle at N/P > or = 3 had a constant size of approximately 90-100 nm. Eventually, acetal-PEG-PAMA/pDNA micelles underwent no precipitation even after long-term storage for more than 1 month at all N/P ratios. The PIC micelles were stable even in the presence of excess polyanions, poly(vinyl sulfate), in contrast to polyplexes based on the PAMA homopolymer, yet this stabilization effect was highly dependent on the N/P ratio to reach a plateau at N/P = 3-4. This character may be attributed to the increased hydrophobicity in the vicinity of the complexed pDNA. Furthermore, the pDNA in the micelle was adequately protected from DNase I attack. The transfection ability of the PIC micelles toward 293 cells was remarkably enhanced with an increasing N/P ratio as high as 25. The zeta-potential of the micelles with a high N/P ratio was an appreciably large positive value, suggesting a noncooperative micelle formation. This deviated micellar composition with an excess cationic nature as well as the presence of free acetal-PEG-PAMA may play a substantial role in the enhanced transfection efficiency of the PIC micelle system in the high N/P ratio (approximately 25) region.