Sequence rearrangement in JC virus DNAs molecularly cloned from immunosuppressed renal transplant patients.

From nonimmunocompromised individuals, we have recently identified a possible archetypal JC virus DNA sequence from which various regulatory sequences of JC virus isolates derived from patients with progressive multifocal leukoencephalopathy (PML) could have evolved. In this study, we analyzed the regulatory sequences of JCV DNAs cloned from urine samples of a PML risk group (renal transplant patients on immunosuppressive therapy). A number of JC virus DNAs were molecularly cloned from virions excreted in the urine of eight patients. Furthermore, fragments containing the regulatory region were amplified by the polymerase chain reaction and subsequently molecularly cloned from cell-associated JC virus excreted in the urine of two patients. The regulatory regions in all clones were analyzed with restriction enzymes, and those in representative clones were sequenced. We found that clones with the archetypal regulatory sequence were predominant in all urine samples, but a few clones carried regulatory sequences that diverged from the archetypal sequence by deletion or duplication. The finding that sequence rearrangement in the archetypal regulatory region occurs in the course of infection in immunosuppressed hosts is consistent with the adaptation hypothesis which has been put forward to explain the divergence of the regulatory regions in PML-derived JC virus isolates.     

Analyses of group III secreted phospholipase A2 transgenic mice reveal potential participation of this enzyme in plasma lipoprotein modification, macrophage foam cell formation, and atherosclerosis

Among the many mammalian secreted phospholipase A2 (sPLA2) enzymes, PLA2G3 (group III secreted phospholipase A2) is unique in that it possesses unusual N- and C-terminal domains and in that its central sPLA2 domain is homologous to bee venom PLA2 rather than to other mammalian sPLA2s. To elucidate the in vivo actions of this atypical sPLA2, we generated transgenic (Tg) mice overexpressing human PLA2G3. Despite marked increases in PLA2 activity and mature 18-kDa PLA2G3 protein in the circulation and tissues, PLA2G3 Tg mice displayed no apparent abnormality up to 9 months of age. However, alterations in plasma lipoproteins were observed in PLA2G3 Tg mice compared with control mice. In vitro incubation of low density (LDL) and high density (HDL) lipoproteins with several sPLA2s showed that phosphatidylcholine was efficiently converted to lysophosphatidylcholine by PLA2G3 as well as by PLA2G5 and PLA2G10, to a lesser extent by PLA2G2F, and only minimally by PLA2G2A and PLA2G2E. PLA2G3-modified LDL, like PLA2G5- or PLA2G10-treated LDL, facilitated the formation of foam cells from macrophages ex vivo. Accumulation of PLA2G3 was detected in the atherosclerotic lesions of humans and apoE-deficient mice. Furthermore, following an atherogenic diet, aortic atherosclerotic lesions were more severe in PLA2G3 Tg mice than in control mice on the apoE-null background, in combination with elevated plasma lysophosphatidylcholine and thromboxane A2 levels. These results collectively suggest a potential functional link between PLA2G3 and atherosclerosis, as has recently been proposed for PLA2G5 and PLA2G10.     

Entry from the cell surface of severe acute respiratory syndrome coronavirus with cleaved S protein as revealed by pseudotype virus bearing cleaved S protein

Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is known to take an endosomal pathway for cell entry; however, it is thought to enter directly from the cell surface when a receptor-bound virion spike (S) protein is affected by trypsin, which induces cleavage of the S protein and activates its fusion potential. This suggests that SARS-CoV bearing a cleaved form of the S protein can enter cells directly from the cell surface without trypsin treatment. To explore this possibility, we introduced a furin-like cleavage sequence in the S protein at amino acids 798 to 801 and found that the mutated S protein was cleaved and induced cell fusion without trypsin treatment when expressed on the cell surface. Furthermore, a pseudotype virus bearing a cleaved S protein was revealed to infect cells in the presence of a lysosomotropic agent as well as a protease inhibitor, both of which are known to block SARS-CoV infection via an endosome, whereas the infection of pseudotypes with an uncleaved, wild-type S protein was blocked by these agents. A heptad repeat peptide, derived from a SARS-CoV S protein that is known to efficiently block infections from the cell surface, blocked the infection by a pseudotype with a cleaved S protein but not that with an uncleaved S protein. Those results indicate that SARS-CoV with a cleaved S protein is able to enter cells directly from the cell surface and agree with the previous observation of the protease-mediated cell surface entry of SARS-CoV.     

Structural Uniformity of Pullulan Produced by Several Strains of Pullularia pullulans

Extracellular polysaccharides produced by 3 strains of Pullularia pullulans were fractionated by treating with cetyl trimethyl ammonium hydroxide into soluble and insoluble fractions, and the structure of the former fraction, i.e., pullulan, was studied. The yield and the ratio of 2 fractions varied widely according to the strains. But the structure of pullulan was found to be uniform irrespective of the strains used. All 3 samples of pullulan gave only glucose on complete acid hydrolysis, and 93~95% maltotriose and 5~7% maltotetraose after isoamylase (pullulanase) action. The ratio of α-1,4- to α-1,6-glucosidic linkages calculated from periodate oxidation data coincided very well with the value expected from the ratio of maltotriose to maltotetraose units. An evidence for the complete absence of branch structure in pullulan was presented from the results of hydrolysis by pullulan 4-glucanohydrolase.     

Comparison of Extracellular Polysaccharide Produced by Pullularia pullulans with Polysaccharides in the Cells and Cell Wall

The purified swep hydrolase appeared homogeneous on SDS disc electrophoresis, and the molecular weights of the enzyme and the subunit were 280,000 and 70,000, respectively. The isoelectric point of this enzyme was pH 4.8.     

Principal Component Analysis Based Feature Extraction Approach to Identify Circulating microRNA Biomarkers

The discovery and characterization of blood-based disease biomarkers are clinically important because blood collection is easy and involves relatively little stress for the patient. However, blood generally reflects not only targeted diseases, but also the whole body status of patients. Thus, the selection of biomarkers may be difficult. In this study, we considered miRNAs as biomarker candidates for several reasons. First, since miRNAs were discovered relatively recently, they have not yet been tested extensively. Second, since the number of miRNAs is relatively limited, selection is expected to be easy. Third, since they are known to play critical roles in a wide range of biological processes, their expression may be disease specific. We applied a newly proposed method to select combinations of miRNAs that discriminate between healthy controls and each of 14 diseases that include 5 cancers. A new feature selection method is based on principal component analysis. Namely this method does not require knowledge of whether each sample was derived from a disease patient or a healthy control. Using this method, we found that hsa-miR-425, hsa-miR-15b, hsa-miR-185, hsa-miR-92a, hsa-miR-140-3p, hsa-miR-320a, hsa-miR-486-5p, hsa-miR-16, hsa-miR-191, hsa-miR-106b, hsa-miR-19b, and hsa-miR-30d were potential biomarkers; combinations of 10 of these miRNAs allowed us to discriminate each disease included in this study from healthy controls. These 12 miRNAs are significantly up- or downregulated in most cancers and other diseases, albeit in a cancer- or disease-specific combinatory manner. Therefore, these 12 miRNAs were also previously reported to be cancer- and disease-related miRNAs. Many disease-specific KEGG pathways were also significantly enriched by target genes of up−/downregulated miRNAs within several combinations of 10 miRNAs among these 12 miRNAs. We also selected miRNAs that could discriminate one disease from another or from healthy controls. These miRNAs were found to be largely overlapped with miRNAs that discriminate each disease from healthy controls.     

Studies on the Pungent Principle of Capsicum

The chemical constitutions of the pungent principle of Capsicum were investigated. These principles are assumed to consist of capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homodihydrocapsaicin and two or more analogues of these materials. Thin-layer chromatography and open tubular gas chromatography showed that the natural pungent mixture contains no cis-isomer of capsaicin. The chemical structure of nordihydrocapsaicin was determined as N-(4-hydroxy-3-methoxybenzyl)-7-methyloctanamide by gas chromatography, infrared spectroscopy, mass spectrometry and nuclear magnetic resonance spectroscopy. In addition, homodihydrocapsaicin was identified as N-(4-hydroxy-3-methoxybenzyl)-9-methyl-decanamide. These identities were also proven by comparison with synthetic samples.