Review of studies that have used knockout mice to assess normal function of prion protein under immunological or pathophysiological stress

Deletion of cellular isoform of prion protein (PrP^C) increases neuronal predisposition to damage by modulating apoptosis and the negative consequences of oxidative stress. In vivo studies have demonstrated that PrP^C‐deficient mice are more prone to seizure, depression, and induction of epilepsy and experience extensive cerebral damage following ischemic challenge or viral infection. In addition, adenovirus‐mediated overexpression of PrP^C reduces brain damage in rat models of cerebral ischemia. In experimental autoimmune encephalomyelitis, PrP^C‐deficient mice reportedly have a more aggressive disease onset and less clinical improvement during the chronic phase than wild‐type mice mice. In mice given oral dextran sulfate, PrP^C has a potential protective role against inflammatory bowel disease. PrP^C‐deficient mice demonstrate significantly greater increases in blood glucose concentrations after intraperitoneal injection of glucose than wild‐type mice. Further in vivo challenges to PrP gene‐deficient models and conditional knockout models with siRNA and in vivo administration of PrP‐ligating agents may assist in refining knowledge of the lymphoid function of PrP^C and predicting the effects of anti‐PrP treatment on the immune system. Together, these findings indicate that PrP^C may have multiple neuroprotective and anti‐inflammatory roles, which explains why this protein is so widely expressed.     

Biogenesis of cytoplasmic lipid droplets: from the lipid ester globule in the membrane to the visible structure

The cytoplasmic lipid droplet (CLD) and very low-density lipoprotein are generated from the lipid ester synthesized in the endoplasmic reticulum. The lipid ester deposited between the two membrane leaflets is supposed to bulge toward the cytoplasm to make a nascent CLD, but its size must be below the resolution limit of conventional techniques and the detectable CLD should only form after acquisition of additional lipid esters. The CLD is different from vesicular organelles in that the internal content is highly hydrophobic and the shape is invariably spherical. Due to its unique characteristics, quantitative discordance between the surface and the volume may occur in the growth and/or involution processes of the CLD. The possibility that these processes may give rise to the structural and functional diversities of the CLD is discussed.     

Spectroscopic diagnosis of chronic fatigue syndrome by visible and near-infrared spectroscopy in serum samples

To investigate visible and near-infrared (Vis-NIR) spectroscopy enabling chronic fatigue syndrome (CFS) diagnosis, we subjected sera from CFS patients as well as healthy donors to Vis-NIR spectroscopy. Vis-NIR spectra in the 600-1100 nm region for sera from 77 CFS patients and 71 healthy donors were subjected to principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA) to develop multivariate models to discriminate between CFS patients and healthy donors. The model was further assessed by the prediction of 99 masked other determinations (54 in the healthy group and 45 in the CFS patient group). The PCA model predicted successful discrimination of the masked samples. The SIMCA model predicted 54 of 54 (100%) healthy donors and 42 of 45 (93.3%) CFS patients of Vis-NIR spectra from masked serum samples correctly. These results suggest that Vis-NIR spectroscopy for sera combined with chemometrics analysis could provide a promising tool to objectively diagnose CFS.     

Invasion assay of Listeria monocytogenes using Vero and Caco-2 cells

The invasion ability of Listeria monocytogenes into cultured cells has been used to evaluate its pathogenicity. In this study, invasive ability was investigated using Vero and Caco-2 cell lines. The form of invasion showed no morphological differences between both cell lines inoculated with L. monocytogenes L89-H2 or L96-23C1 strains when double fluorescence stained with rhodamine and FITC or with Giemsa staining. Recovery count and recovery rate of L. monocytogenes from Vero cells was related to the number of inoculated bacteria (2 x 10(5) to 2 x 10(7)/ml) in a bell-shape pattern, though the relationship was unclear in Caco-2 cells. Recovery rate of L. monocytogenes was higher in Vero cells than Caco-2 cells at a multiplicity of infection (MOI) 10, though the rates in both cells showed different stable stages over a considerably wide range of MOI. The recovery rate of all five L. monocytogenes strains from listeriosis patients was 15% at MOI 10 from infected Vero cells, while meat-derived strains showed variable rates regardless of the serovar. These results suggest that the Vero cell line is suitable for an invasion assay and that a recovery rate of 15% may be the critical limit for the expression of pathogenicity in the host.     

Fusion of Doppel to octapeptide repeat and N-terminal half of hydrophobic region of prion protein confers resistance to serum deprivation

Our previous studies have shown an essential role played by the octapeptide repeat region (OR) and the N-terminal half of hydrophobic region (HR) in the anti-apoptotic activity of prion protein (PrP). As PrP-like protein Doppel (Dpl), which structurally resembles an N-terminally truncated PrP, did not show any anti-apoptotic activity, we examined apoptosis of HpL3-4 cells expressing Dpl fused to various lengths of the N-terminal region of PrP to investigate whether the PrP/Dpl fusion proteins retain anti-apoptotic function. HpL3-4 cells expressing Dpl fused to PrP(1-124) with the OR and N-terminal half of HR of PrP showed anti-apoptotic function, whereas Dpl fused to PrP(1-95) with OR did not rescue cells from apoptotic cell death induced by serum deprivation. These results indicate that the OR and N-terminal half of HR of PrP retains anti-apoptotic activity similar to full-length PrP.     

Simultaneous analysis of human plasma catecholamines by high-performance liquid chromatography with a reversed-phase triacontylsilyl silica column

The clinical importance of simultaneous analysis of 3,4-dihydroxyphenylglycol with other human plasma catecholamines has been investigated to better understand the sympathetic nervous system. However, previous reports have had analytical difficulties with both resolution and extraction. The current study uses a reversed-phase triacontylsilyl silica (C30) column under the mobile phase condition without ion-pair reagents to separate catecholamines and their metabolites, with above 91% recoveries for intra-assay, above 85% for inter-assay, and less than 10% (n=5) coefficient of variation. Lower detection limits (S/N=4) and quantification limits (S/N=6) were 40 and 100 pg/mL for norepinephrine, 3,4-dihydroxyphenylglycol, and 3,4-dihydroxyphenylalanine, 10 and 20 pg/mL for epinephrine, 10 and 40 pg/mL for dopamine. Linear ranges were from 40 to 5000 pg/mL for norepinephrine and 3,4-dihydroxyphenylalanine, from 100 to 5000 pg/mL for 3,4-dihydroxyphenylglycol, and from 10 to 2000 pg/mL for epinephrine and dopamine. The C30 column may prove clinically useful, as it provides a convenient and simultaneous method of evaluation of human plasma catecholamines.     

Stereoselective Synthesis of Optically Active Dictyopterenes A and B and Their Geometrical Isomers

Optically active dictyopterenes A and B and their geometrical isomers were stereoselectively synthesized by condensation of acrolein with carboethoxymethyl dimethylsulfonium bromide and by the Wittig reaction between (+)-2-vinylcyclopropylcarbaldehyde, which was derived from partially resolved (+)-(1,S, 2R)-2-vinylcyclopropanecarboxylic acid, and phosphonium salts in liquid-solid two-phase systems using crown ethers.     

Volatile C6-Aldehyde Formation via Hydroperoxides from C18-Unsaturated Fatty Acids in Etiolated Alfalfa and Cucumber Seedlings

1. Etiolated seedlings of alfalfa and cucumber evolved n-hexanal from linoleic acid and cis-3-hexenal and trans-2-hexenal from linolenic acid when they were homogenized.

2. The activities for n-hexanal formation from linoleic acid, lipoxygenase and hydro-peroxide lyase were maximum in dry seeds and 1~2 day-old etiolated seedlings of alfalfa, and in 6~7 day-old etiolated seedlings of cucumber.

3. n-Hexanal was produced from linoleic acid and 13-hydroperoxylinoleic acid by the crude extracts of etiolated alfalfa and cucumber seedlings. cis-3-Hexenal and trans-2-hexenal were produced from linolenic acid and 13-hydroperoxylinolenic acid by the crude extracts of etiolated alfalfa and cucumber seedlings. But these extracts, particulariy cucumber one, showed a high isomerizing activity from cis-3-hexenal to trans-2-hexenal.

4. When the C₈-aldehydes were produced from linoleic acid and linolenic acid by the crude extracts, formation of hydroperoxides of these C₁₈-fatty acids was observed.

5. When 9-hydroperoxylinoleic acid was used as a substrate, trans-2-nonenal was produced by the cucumber homogenate but not by the alfalfa homogenate.

6. As the enzymes concerned with C₆-aldehyde formation, lipoxygenase was partially purified from alfalfa and cucumber seedlings and hydroperoxide lyase, from cucumber seedlings. Lipoxygenase was found in a soluble fraction, but hydroperoxide lyase was in a membrane bound form. Alfalfa lipoxygenase catalyzed formation of 9- and 13-hydroperoxylinoleic acid (35: 65) from linoleic acid and cucumber one, mainly 13-hydroperoxylinoleic acid formation. Alfalfa hydroperoxide lyase catalyzed n-hexanal formation from 13-hydroperoxylinoleic acid, but cucumber one catalyzed formation of n-hexanal and trans-2-nonenal from 13- and 9-hydroperoxylinoleic acid, respectively.

7. From the above results, the biosynthetic pathway for C₆-aldehyde formation in etiolated alfalfa and cucumber seedlings is established that C₆-aldehydes (n-hexanal, cis-3-hexenal and trans-2-hexenal) are produced from linoleic acid and linolenic acid via their 13-hydroperoxides by lipoxygenase and hydroperoxide lyase.     

Leaf Alcohol

Successful oxidations with chromic acid-sulfuric acid mixture of synthetic isomeric hex-2-en-l-ols to give trans-hex-2-en-l-al (leaf aldehyde) and the failure of those of synthetic isomeric hex-3-en-l-ols pointed out the probable contamination of the 2-enol isomer in the earlier “leaf alcohol preparation” of plant origin, which might lead the workers to an erroneous conclusion.

In support of the present worker’s opinion, trans-hex-2-en-l-ol was actually isolated from fresh tea leaves and identified in the present investigation and a mechanistic rational was invoked to account for the results of oxidations of isomeric n-hexenols.