An enzymatically stable kyotorphin analog induces pain in subattomol doses

Intraplantar injection of the enzymatically stable, N-methylated kyotorphin analog Tyr(NMe)-Arg-OH produced marked and sharp nociceptive flexor responses in a dose-dependent manner. A significant response was observed with this compound at a dose of 0. 01 amol (6000 molecules). Tyr(NMe)-Arg-OH-nociception was completely blocked by the kyotorphin antagonist leucyl-arginine and its enzymatically stable, N-methylated analog, as well as by CP-99994, a specific neurokinin 1 antagonist. These findings suggest that the nociceptive effect produced by Tyr(NMe)-Arg-OH in subattomol doses occurs via specific interaction with the kyotorphin receptor and that the extraordinary potency observed may result from amplification through local substance P release.     

Olfactory discriminating ability of lacustrine sockeye and masu salmon in various freshwaters

In order to study the olfactory discriminating ability of lacustrine sockeye salmon (Oncorhynchus nerka) and masu salmon (O. masou), the integrated olfactory nerve response to various freshwaters was recorded by electrophysiological techniques. In both species independent of sex and gonadal maturity, each freshwater caused a different olfactory response. Source and effluent waters of the culture pond at Toya Lake Station (the source and culture pond waters) evoked the minimum and maximum response magnitude, respectively. In cross-adaptation experiments, the culture pond water abolished all secondary responses to other freshwaters, and no freshwater abolished the secondary response to the culture pond water. The concentration response study revealed that the minimum concentration (threshold) to induce response to the culture pond water after adaptation to Lake Toya water was between 0.1 and 1.0%. The present study indicates that the olfactory organ of lacustrine salmonids may discriminate different intensities of various freshwater odors.     

The eosinophil peroxidase gene forms a cluster with the genes for myeloperoxidase and lactoperoxidase on human chromosome 17

Eosinophil peroxidase (EPX) is one of a family of mammalian peroxidases that includes myeloperoxidase (MPO), lactoperoxidase (LPO), and thyroid peroxidase (TPO). Here we show that the human EPX gene maps to chromosome 17q23.1, which localizes 34 kb from the LPO and MPO genes. Our results demonstrate that the EPX, LPO, and MPO genes form a cluster on human chromosome 17.     

Effects of pH and light on the storage stability of the purple pigment, hordeumin, from uncooked barley bran fermented broth

The pigment retention rate of hordeumin was higher than that of two standard anthocyanidins, cyanidin and delphinidin, when hordeumin and anthocyanidins were dissolved in Walpole buffer (pH 1.0) and stored. Moreover, when pigment solutions were stored at 15 degrees C under light irradiation, the pigment retention rate of the hordeumin solution became higher than those of standard anthocyanidins (2 to 10 times) as the storage period increased. Comparing various pH buffers (MacIlvaine buffer, pH 2.2 to 7.0), the pigment retention rate of hordeumin at pH 5.0 was highest. Furthermore, the half-life of hordeumin at pH 5.0 was increased from 9 days to 17.5 days when nitrogen gas was bubbled into the hordeumin solution. We considered that the storage stability of hordeumin is higher than standard anthocyanidins because hordeumin is a complex with anthocyanin, tannin, and protein.     

Development of clover yellow vein virus as an efficient, stable gene-expression system for legume species

A highly infectious cDNA clone of clover yellow vein virus (pClYVV) was tested as a viral vector, especially for legume species. The genes for green fluorescent protein (GFP) and soybean glutamine synthetase (GS) were inserted between the genes for P1 and HC-Pro on pClYVV to create three recombinant plasmids: pClYVV-GFP, pClYVV-GFP-GS, and pClYVV-GFP:GS. In the former two constructs all the junctions between the inserted proteins contained the sequences of protease cleavage recognition sites, whereas the third construct expressed a fusion of GFP and GS. Western blot analyses showed that GFP and GS appeared to have been precisely excised from the viral polyprotein with the viral proteases (P1 and NIa). Under UV irradiation, green fluorescence was detected in infected broad bean, kidney bean, and soybean plants. The stability of the constructs in the symptomatic tissues was confirmed by RT-PCR and Western blot analyses. The plants expressing GS together with GFP became tolerant to the herbicide glufosinate, and flowered early. As the GS gene, one of the nodulin genes for nitrogen fixation, is expressed in legume species, this system will be useful for examining the function of genes important to legume plants.     

Heterogeneity of high density lipoprotein generated by ABCA1 and ABCA7

The assembly of HDL by helical apolipoprotein and cellular lipid was studied using HEK293 cells to which ecdysone-inducible human ABCA1 or human ABCA7 was transfected. Expression of both ABCA1 and ABCA7 was induced linearly proportional to ponasterone A concentration in the medium. In the experimental conditions used, the ABC protein expression levels limited the rate of lipid release when the apolipoprotein concentration was high, and the apolipoprotein concentration was rate-limiting when the ABC protein expression levels were high. When ABCA1 expression increased in conditions in which it was rate-limiting, relative cholesterol content to phospholipid increased in the HDL produced. In contrast, it was constant when ABCA7 expression increased. To investigate the background mechanism, the HDL particles were analyzed by density gradient ultracentrifugation and high performance lipid chromatography. The ABCA1-mediated reaction produced two distinct HDLs, large cholesterol-rich and small cholesterol-poor particles, and the ABCA7-mediated reaction generated mostly small cholesterol-poor particles. The increase of HDL assembly with the increase of ABCA1 expression was predominant in large cholesterol-rich particles, whereas only small cholesterol-poor HDL increased as ABCA7 expression increased. We conclude that ABCA1 generates cholesterol-rich and cholesterol-poor HDL and that the former is more prominently dependent on the increase of ABCA1 expression. ABCA7 produces this HDL subfraction only as a very minor component.     

Translocation of a 190-kb mitochondrial fragment into rice chromosome 12 followed by the integration of four retrotransposons

A 190-kb mitochondrial DNA sequence interrupted by seven foreign DNA segments was identified in rice chromosome 12. This fragment is the largest mitochondrial fragment translocated into the rice nuclear genome. The sequence is composed of a 190-kb segment of mitochondrial origin corresponding to 38.79% of the mitochondrial genome, 45 kb comprising four segments of retrotransposon origin, and 13 kb comprising three segments of unknown origin. The 190-kb sequence shows more than 99.68% similarity to the current mitochondrial sequence, suggesting that its integration into the nucleus was quite recent. Several sequences in the 190-kb segment have been rearranged relative to the current mitochondrial sequence, suggesting that the past and present arrangements of the mitochondrial genome differ. The four retrotransposons show no mutual sequence similarity and are integrated into different locations, suggesting that their integration events were independent, frequent, and quite recent. A fragment of the mitochondrial genome present in the nuclear genome, such as the 248-kb sequence characterized in this study, is a good relic with which to investigate the past mitochondrial genome structure and the behavior of independent retrotransposons during evolution.     

Cloning and functional analysis of the rhesus macaque ABCG2 gene. Forced expression confers an SP phenotype among hematopoietic stem cell progeny in vivo

Hematopoietic cells can be highly enriched for repopulating ability based upon the efflux of the fluorescent Hoechst 33342 dye by sorting for SP (side population) cells, a phenotype attributed to expression of ABCG2, a member of the ABC transporter superfamily. Intriguingly, murine studies suggest that forced ABCG2 expression prevents hematopoietic differentiation. We cloned the full-length rhesus ABCG2 and introduced it into a retroviral vector. ABCG2-transduced human peripheral blood progenitor cells (PBPCs) acquired the SP phenotype but showed significantly reduced growth compared with control. Two rhesus macaques received autologous PBPCs split for transduction with the ABCG2 or control vectors. Marking levels were similar between fractions with no discrepancy between bone marrow and peripheral blood marking. Analysis for the SP phenotype among bone marrow and mature blood populations confirmed ABCG2 expression at levels predicted by vector copy number long term, demonstrating no block to differentiation in the large animal. In vitro studies showed selective protection against mitoxantrone among ABCG2-transduced rhesus PBPCs. Our results confirm the existence of rhesus ABCG2, establish its importance in conferring the SP phenotype, suggest no detrimental effect of its overexpression upon differentiation in vivo, and imply a potential role for its overexpression as an in vivo selection strategy for gene therapy applications.