Mouse-Hamster Chimeric Prion Protein (PrP) Devoid of N-Terminal Residues 23-88 Restores Susceptibility to 22L Prions,but Not to RML Prions in PrP-Knockout Mice

Prion infection induces conformational conversion of the normal prion protein PrP^C, into the pathogenic isoform PrP^Sc, in prion diseases. It has been shown that PrP-knockout (Prnp^0/0) mice transgenically reconstituted with a mouse-hamster chimeric PrP lacking N-terminal residues 23-88, or Tg(MHM2Δ23-88)/Prnp^0/0 mice, neither developed the disease nor accumulated MHM2^ScΔ23-88 in their brains after inoculation with RML prions. In contrast, RML-inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice developed the disease with abundant accumulation of MHM2^ScΔ23-88 in their brains. These results indicate that MHM2Δ23-88 itself might either lose or greatly reduce the converting capacity to MHM2^ScΔ23-88, and that the co-expressing wild-type PrP^C can stimulate the conversion of MHM2Δ23-88 to MHM2^ScΔ23-88 in trans. In the present study, we confirmed that Tg(MHM2Δ23-88)/Prnp^0/0 mice remained resistant to RML prions for up to 730 days after inoculation. However, we found that Tg(MHM2Δ23-88)/Prnp^0/0 mice were susceptible to 22L prions, developing the disease with prolonged incubation times and accumulating MHM2^ScΔ23-88 in their brains. We also found accelerated conversion of MHM2Δ23-88 into MHM2^ScΔ23-88 in the brains of RML- and 22L-inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice. However, wild-type PrP^Sc accumulated less in the brains of these inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice, compared with RML- and 22L-inoculated Prnp^0/+ mice. These results show that MHM2Δ23-88 itself can convert into MHM2^ScΔ23-88 without the help of the trans-acting PrP^C, and that, irrespective of prion strains inoculated, the co-expressing wild-type PrP^C stimulates the conversion of MHM2Δ23-88 into MHM2^ScΔ23-88, but to the contrary, the co-expressing MHM2Δ23-88 disturbs the conversion of wild-type PrP^C into PrP^Sc.     

Production of biologically active human thioredoxin 1 protein in lettuce chloroplasts

The production of human therapeutic proteins in plants provides opportunities for low-cost production, and minimizes the risk of contamination from potential human pathogens. Chloroplast genetic engineering is a particularly promising strategy, because plant chloroplasts can produce large amounts of foreign target proteins. Oxidative stress is a key factor in various human diseases. Human thioredoxin 1 (hTrx1) is a stress-induced protein that functions as an antioxidant against oxidative stress, and overexpression of hTrx1 has been shown to suppress various diseases in mice. Therefore, hTrx1 is a prospective candidate as a new human therapeutic protein. We created transplastomic lettuce expressing hTrx1 under the control of the psbA promoter. Transplastomic plants grew normally and were fertile. The hTrx1 protein accumulated to approximately 1% of total soluble protein in mature leaves. The hTrx1 protein purified from lettuce leaves was functionally active, and reduced insulin disulfides. The purified protein protected mouse insulinoma line 6 cells from damage by hydrogen peroxide, as reported previously for a recombinant hTrx1 expressed in Escherichia coli. This is the first report of expression of the biologically active hTrx1 protein in plant chloroplasts. This research opens up possibilities for plant-based production of hTrx1. Considering that this expression host is an edible crop plant, this transplastomic lettuce may be suitable for oral delivery of hTrx1.     

A case of hyperlipidemia with homozygous apolipoprotein E5 (Glu3-->Lys)

In this study, we present clinical feature of a novel case with homozygous apolipoprotein (apo) E5.The patient was a 53-year-old Japanese woman. She was from a small island off the coast of Kagoshima Prefecture, Japan. Her parents were first degree cousins. No corneal opacification, xanthomatosis, lymphadenopathy, or hepatosplenomegaly was observed. There have been no signs of clinically overt atherosclerosis to date. Her serum total cholesterol, triglycerides (TG) and high-density lipoprotein (HDL)-cholesterol levels were 11.6, 6.1 and 1.2 mmol/l, respectively, and apo A-I, A-II, B, C-II, C-III and E levels were 121, 34.8, 269, 10.4, 25.7 and 10.3 mg/dl, respectively. Serum lipoprotein profile analyzed by agarose gel electrophoresis and differential staining revealed markedly increased cholesterol and TG in both beta and prebeta-migrated lipoproteins, whereas alpha-migrated lipoprotein showed decreased cholesterol. Her apo E isoform analyzed by isoelectric focusing (IEF) was found to be homozygous apo E5.Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis of her apo E and lipoprotein lipase (LPL) genes revealed that she had a homozygous apo E (Glu3-->Lys) and heterozygous LPL variant Ser447 to Ter. Her son and daughter, both of whom had hyperlipidemia, were found to have apo E3/5 phenotype. Direct sequencing analysis of her apo E gene confirmed a homozygous one nucleotide change: G to A at nucleotide position of 2836 in the exon 3, resulting in Glu3-->Lys mutation.This is the first report of lipids and lipoprotein profiles in patients with homozygous apo E5 (Glu3-->Lys).     

Upregulation of proteinase-activated receptors and hypercontractile responses precede development of arterial lesions after balloon injury

Thrombin and other proteinases exert vascular effects by activating the proteinase-activated receptors (PARs). The expression of PARs has been shown to be upregulated after balloon injury and in human arteriosclerosis. However, the relationship between the receptor upregulation and the alteration of vasomotor function remains to be elucidated. We herein demonstrated that the contractile responses to the PAR-1 and PAR-2 agonist were markedly enhanced in the rabbit femoral arteries after balloon injury. Neointimal thickening was established 4 wk after the injury. No histological change was observed in the sham operation, where the saphenous artery was ligated without any balloon injury. The contractile response to K(+) depolarization was significantly attenuated 1 wk after the injury and then partly recovered after 4 wk. Thrombin, PAR-1-activating peptide, trypsin, and PAR-2-activating peptide induced no significant contraction in the control. All these stimulants induced enhanced responses 1 wk after balloon injury. Such enhanced responses were seen 4 wk after the injury, except for thrombin. There was no change in the Ca(2+) sensitivity of the contractile apparatus as evaluated in the permeabilized preparations. PAR-1-activating peptide (100 mumol/l), but no other stimulants, induced an enhanced contraction in the sham operation. The expression of PAR-1 and PAR-2 slightly increased after the sham operation, whereas it markedly and significantly increased after balloon injury. Our observations suggest that balloon injury induced the receptor upregulation, thereby enhancing the contractile response before the establishment of vascular lesions. The local inflammation associated with the sham operation may also contribute to the receptor upregulation.     

Quantitative Identification of Mutant Alleles Derived from Lung Cancer in Plasma Cell-Free DNA via Anomaly Detection Using Deep Sequencing Data

The detection of rare mutants using next generation sequencing has considerable potential for diagnostic applications. Detecting circulating tumor DNA is the foremost application of this approach. The major obstacle to its use is the high read error rate of next-generation sequencers. Rather than increasing the accuracy of final sequences, we detected rare mutations using a semiconductor sequencer and a set of anomaly detection criteria based on a statistical model of the read error rate at each error position. Statistical models were deduced from sequence data from normal samples. We detected epidermal growth factor receptor (EGFR) mutations in the plasma DNA of lung cancer patients. Single-pass deep sequencing (>100,000 reads) was able to detect one activating mutant allele in 10,000 normal alleles. We confirmed the method using 22 prospective and 155 retrospective samples, mostly consisting of DNA purified from plasma. A temporal analysis suggested potential applications for disease management and for therapeutic decision making to select epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI).     

Human corneal epithelial cells respond to ocular-pathogenic, but not to nonpathogenic-flagellin

In this study, we investigated the expression of TLR5 in human corneal epithelial cells (CEC), and the functional outcome of TLR5 triggering by flagellins of pathogenic- and nonpathogenic bacteria. Flagellins derived from Pseudomonas aeruginosa, Salmonella typhimurium, Serratia marcescense or Bacillus subtilis were used. The TLR5 protein and TLR5 specific mRNA expression was evident on human CEC. In human corneal epithelium tissues, TLR5 protein was detected at the basal and wing cells of the tissues. Ocular pathogenic bacteria, namely P. aeruginosa and S. marcescense, derived flagellin induced the significantly increased level of gene activation and IL-6 and IL-8 production. In contrast, ocular nonpathogenic S. typhimurium- and B. subtilis-derived flagellin induced neither the gene activation nor the increased production of IL-6 and IL-8 in human CEC. Human CEC would respond only to flagellin derived of ocular pathogenic bacteria, but not to those derived of ocular nonpathogenic bacteria, to generate pro-inflammatory cytokines.     

Pyramiding of male-sterile genes in <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> D. Don with the aid of closely linked markers

Gene pyramiding is a breeding method used to combine multiple useful genes. Although several genes have been pyramided in certain crops, gene pyramiding has not previously been applied to forest trees. In this study, we used the markers closely linked to the two male-sterile genes MS1 and MS2 for the effective development of individuals doubly heterozygous for these two genes. This is the first example of gene pyramiding through marker-assisted selection (MAS) in forest trees. The markers gSNP06239, which is closely linked to the MS1 gene, and estSNP00695, which is closely linked to MS2, were used in MAS. On the basis of the linkage phase between the markers and male-sterile loci, we selected five F₁ individuals (S3-64 from Shindai-3 × Kamikiri-31, S3-70 from Shindai-3 × Kamikiri-38, S3-77 from Shindai-3 × Kamikiri-47, S1-22 from Shindai-1 × Nakakubiki-4, and S1-56 from Shindai-1 × Setsugai-20) as parents for artificial crossing. The 268 seedlings obtained from six artificial cross combinations were used in this study. Chi-squared tests showed no significant deviation from the expected Mendelian ratios of genotypes, indicating that MAS using markers closely linked to the male-sterile genes worked very well. Fifteen individuals that showed unexpected genotypes were probably recombinants, because the map distances between the male-sterile locus and the DNA markers were 4.1 cM (gSNP06239 to MS1) and 6.9 cM (estSNP00695 to MS2). Development of markers more closely linked to the male-sterile loci will facilitate precise gene pyramiding in the future.     

Geometrical Isomers of Monohydroperoxides Formed by Autoxidation of Methyl Linoleate

Isomeric monohydroperoxides produced from autoxidized methyl linoleate were separated into two geometrical isomers (cis-trans and trans-trans) by silver nitrate TLC. Purified monohydroperoxides were converted into hydroxy octadecadienoates. Trimethylsilyl (TMS) derivatives of these compounds (four components) were separated into three peaks in the gas chromatogram; the mixture of 9-hydroxy-cis,trans-isomer and 13-hydroxy-cis,trans-isomer, 9-hydroxy-trans,trans-isomer and 13-hydroxy-trans,trans-isomer. The trans-trans isomers became more dominant than the cis-trans isomers in the later stage of autoxidation and with the rise of temperature. At the degradation of monohydroperoxides, the decrease of trans- trans isomers was apparently slower than that of cis-trans isomers. It is proposed that cis,trans isomerization of monohydroperoxides takes place at the process of autoxidation of methyl linoleate.     

Single Quantum Dot Tracking Reveals that an Individual Multivalent HIV-1 Tat Protein Transduction Domain Can Activate Machinery for Lateral Transport and Endocytosis

The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP''s behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell''s lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines.