Reduction in the IgE reactivity of Pacific mackerel parvalbumin by mutations at Ca2+-binding sites

ABSTRACT:   Parvalbumin is a sarcoplasmic Ca²⁺-binding protein of 12 kDa and represents the major fish allergen. Several peptide segments are identified as immunoglobulin E (IgE)-binding epitopes of cod parvalbumin. However, carp parvalbumin (Cyp c 1) shows a markedly reduced IgE-binding ability upon depletion of Ca²⁺, suggesting the importance of conformational epitopes associated with Ca²⁺-chelating. In this study, the IgE reactivity of Pacific mackerel Scomber japonicus parvalbumin (Sco j 1) was demonstrated to be markedly reduced (60–100% reduction) by Ca²⁺-depletion, similar to Cyp c 1. Three Sco j 1 mutants (D51A, D90A, D51/90A), with modifications in either one or both of the two Ca²⁺-binding sites, were then constructed by site-directed mutagenesis, followed by expression in Escherichia coli , and evaluated for their IgE reactivity. Interestingly, the double mutant (D51/90A), probably devoid of Ca²⁺-binding capacity, exhibited a significantly reduced IgE reactivity (equivalent to 0.0–7.5% of the IgE reactivity of natural Sco j 1). The results suggest that the IgE-binding ability of Sco j 1 largely depends on the solid conformation mediated by Ca²⁺-chelating, and that the hypoallergenic D51/90A will be a useful tool for the specific immunotherapy of fish allergy.     

An expanded palette of genetically encoded Ca²⁺ indicators

Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in response to changes in calcium ion (Ca(2+)) concentration are powerful tools for visualizing intracellular signaling activity. However, despite a decade of availability, the palette of single FP-based Ca(2+) indicators has remained limited to a single green hue. We have expanded this palette by developing blue, improved green, and red intensiometric indicators, as well as an emission ratiometric indicator with an 11,000% ratio change. This series enables improved single-color Ca(2+) imaging in neurons and transgenic Caenorhabditis elegans. In HeLa cells, Ca(2+) was imaged in three subcellular compartments, and, in conjunction with a cyan FP-yellow FP-based indicator, Ca(2+) and adenosine 5'-triphosphate were simultaneously imaged. This palette of indicators paints the way to a colorful new era of Ca(2+) imaging.     

Reactivity of lignin with different composition of aromatic syringyl/guaiacyl structures and erythro/threo side chain structures in β-O-4 type during alkaline delignification: as a basis for the different degradability of hardwood and softwood lignin

The reactivity of lignin during alkaline delignification was quantitatively investigated focusing on the effect of the structural differences between syringyl and guaiacyl aromatic nuclei and between erythro and threo in the side chain of β-O-4 type lignin substructure on the β-O-4 bond cleavage rate. It was known that the ratio of this reaction rate of the erythro to threo isomers of the dimeric β-O-4 type lignin model compound with two guaiacyl aromatic nuclei was ca. 4. However, the presence of a syringyl nucleus strongly influenced the rate, and the ratio of the syringyl type analogue was in the range between 2.7 and 8.0 depending on the reaction temperature. The effect of syringyl nucleus on the enhancement of the reaction rate appeared to be greater when the syringyl nucleus consists of the cleaving ether bond rather than being a member of the carbon framework.     

Effect of iron-quercetin complex on reduction of nitrite in in vitro and in vivo systems

This study investigated whether reducing agents such as quercetin and iron(II) facilitate formation of nitric oxide (NO) gas from orally ingested nitrite in an vivo study. When 3 mg/kg Na (15)NO2 was orally administered to rats with or without iron(II) or quercetin, Hb (15)NO, which is indicative of systemic (15)NO, was detected in the blood, with the maximum blood concentration of Hb (15)NO at 15 min after nitrite or nitrite plus quercetin treatment, whereas after administration of nitrite plus iron(II) or nitrite plus iron(II) and quercetin, the time was shortened to 10 min. Interestingly, iron(II), quercetin, or iron(II) plus quercetin did not affect the total amount of Hb (15)NO generated from orally administered Na (15)NO2. However, the systemic nitrite concentration was significantly decreased in the presence of iron(II) or iron(II) plus quercetin. These results may indicate that iron(II) is critical to the generation of NO gas from nitrite, whereas quercetin contributed little under the in vivo experimental conditions.     

Leaf margin phenotype-specific restriction-site-associated DNA-derived markers for pineapple (Ananas comosus L.)

To explore genome-wide DNA polymorphisms and identify DNA markers for leaf margin phenotypes, a restriction-site-associated DNA sequencing analysis was employed to analyze three bulked DNAs of F1 progeny from a cross between a ‘piping-leaf-type’ cultivar, ‘Yugafu’, and a ‘spiny-tip-leaf-type’ variety, ‘Yonekura’. The parents were both Ananas comosus var. comosus. From the analysis, piping-leaf and spiny-tip-leaf gene-specific restriction-site-associated DNA sequencing tags were obtained and designated as PLSTs and STLSTs, respectively. The five PLSTs and two STSLTs were successfully converted to cleaved amplified polymorphic sequence (CAPS) or simple sequence repeat (SSR) markers using the sequence differences between alleles. Based on the genotyping of the F1 with two SSR and three CAPS markers, the five PLST markers were mapped in the vicinity of the P locus, with the closest marker, PLST1_SSR, being located 1.5 cM from the P locus. The two CAPS markers from STLST1 and STLST3 perfectly assessed the ‘spiny-leaf type’ as homozygotes of the recessive s allele of the S gene. The recombination value between the S locus and STLST loci was 2.4, and STLSTs were located 2.2 cM from the S locus. SSR and CAPS markers are applicable to marker-assisted selection of leaf margin phenotypes in pineapple breeding.     

Oolong tea theasinensins attenuate cyclooxygenase-2 expression in lipopolysaccharide (LPS)-activated mouse macrophages: structure-activity relationship and molecular mechanisms

Oolong tea theasinensins are a group of tea polyphenols different from green tea catechins and black tea theaflavins. The present study reports the inhibitory effects of oolong tea theasinensins on the expression of cyclooxygenase-2 (COX-2) and underlying molecular mechanisms in lipopolysaccharide (LPS)-activated murine macrophage RAW264 cells. The structure-activity data revealed that the galloyl moiety of theasinensins played an important role in the inhibitory actions. Theasinensin A, a more potent inhibitor, caused a dose-dependent inhibition of mRNA, protein, and promoter activity of COX-2. An electrophoretic mobility shift assay (EMSA) revealed that theasinensin A reduced the complex of NF-κB- and AP-1-DNA in the promoter of COX-2. Signaling analysis demonstrated that theasinensin A attenuated IκB-α degradation, nuclear p65 accumulation, and c-Jun phosphorylation. Furthermore, theasinensin A suppressed the phosphorylation of MAPKs, IκB kinase α/β (IKKα/β), and TGF-β activated kinase (TAK1). These data demonstrated that the down-regulation of TAK1-mediated MAPKs and NF-κB signaling pathways might be involved in the inhibition of COX-2 expression by theasinensin A. These findings provide the first molecular basis for the anti-inflammatory properties of oolong tea theasinensins.     

Repeated applications of farmyard manure enhance resistance and resilience of soil biological functions against soil disinfection

The effect of repeated application of farmyard manure (FYM) on resistance and resilience of some of soil biological functions was compared between two soils [chemically fertilized soil (CF soil) and chemically and organically amended soil (CF+FYM-soil)]. The impact of chloropicrin and metam sodium disinfection on organic matter (glucose and chitin) decomposition activity and number of ammonia oxidizing bacteria in the soils was measured periodically from just after disinfection to evaluate stability of soil biological functions to soil disinfection. Glucose and chitin decomposition activity and number of ammonia oxidizing bacteria was suppressed by soil disinfection more seriously in the CF soil than in the CF+FYM soil. The decomposition rates recovered in the disinfected CF+FYM soil 2 to 12 weeks after disinfection, but not in the CF soil during 12 weeks incubation. These results demonstrated that soil resistance and resilience of the selected biological functions may be higher in the CF+ FYM soil than in the CF soil. The ratios of fungal and bacterial contribution to glucose decomposition activity were evaluated by the substrate-induced respiration method. Chloropicrin and metam sodium disinfection decreased fungal contribution throughout the incubation period (12 weeks) in the CF soil. A similar tendency was observed in the CF+FYM soil, but the fungal contribution tended to recover more rapidly in the CF+FYM soil than in the CF soil. Repeated applications of FYM may be an effective way to enhance soil functional stability.     

Importance of AC-rich Element on Pea Phenylalanine Ammonia-Lyase Gene 1 Promoter for Expression Induced by Nonpathogenic Attack

Regulatory elements in the promoter of phenylalanine ammonia-lyase gene 1 of pea (PSPAL1) in response to nonpathogenic attack were identified by in vivo footprinting analysis. The footprints determined AC-rich sequences, Box-I and Box-II, that were conserved at similar positions in the phenylpropanoid gene promoters from several plants. To reveal the functions of the AC-rich sequence in nonpathogen-responsiveness, we constructed Box-I-deletion PSPAL1 promoter (dB-1) with GUS reporter gene and transformed it into tobacco plant. The dB-1 had reduced basal expression and a complete loss of nonpathogen-responsiveness. These results indicate the essentiality of Box-I for PSPAL1 activation induced by nonpathogenic attack. Received 27 October 1999/ Accepted in revised form 25 November 1999     

Inhibin secretion in the golden hamster (Mesocricetus auratus) testis during active and inactive states of spermatogenesis induced by the restriction of photoperiod

Gonadal function in the male golden hamster (Mesocricetus auratus) was investigated during exposure to a short photoperiod condition. Within 3 weeks of exposure to the short photoperiod condition, FSH and testosterone in the plasma significantly decreased, and subsequently immunoreactive (ir)-inhibin significantly decreased. Testicular contents of ir-inhibin and testosterone, and pituitary contents of LH and FSH also significantly decreased by 3 weeks with regression of weight of testes, epididymis and seminal vesicles and sperm head count. Circulating LH varied but not significantly. Thereafter, all reproductive parameters and secretion of LH, FSH, ir-inhibin and testosterone gradually recovered after 17 weeks of exposure even though animals continued to be subjected to the short photoperiod condition. Plasma concentrations of inhibin B and inhibin pro-alphaC were detectable and were significantly decreased after 15 weeks of exposure to the short photoperiod, but their levels were still detectable. Immunopositive reaction of inhibin alpha and betaB subunits was found in Sertoli cells and Leydig cells in the regressed testes of animals subjected to short photoperiod as was also seen in animals before exposure to the short photoperiod. Although the spermatogenic cycle was suppressed like prepubertal animals, the present study showed that the testicular recovery, so-called refractoriness, is functionally different from the developing stage of immature animals, especially with regard to inhibin secretion. The present results showed that changes in FSH preceded changes in inhibin during the regression and recovery phases, indicating that FSH is a major regulatory factor of inhibin secretion in male golden hamsters. The present study also demonstrated that regressed testes still secrete a small amount of bioactive inhibin during exposure to a short-photoperiod condition.     

Effect of vasoactive intestinal peptide on the release of growth hormone in perifused pituitary and hypothalamus of the goat

The effect of vasoactive intestinal peptide (VIP) on the release of growth hormone (GH) from the adenohypophysis of the goat was studied in vitro using the perifusion system. Two perifusion systems were employed to differentiate potential direct effects of VIP on the pituitary from indirect effects mediated through the hypothalamus. The first perifusion system used single chambers housing only pituitary fragments. The second system used two chambers in tandem, one containing hypothalamus and the second the pituitary fragments, the flow passing through the hypothalamic chamber first. VIP (10(-6), 10(-7), 10(-8)M) stimulated significant GH release in both perifusion systems (P less than 0.05, P less than 0.01) in a concentration related fashion. The quantity of GH release induced by the 10(-9)M and 10(-10)M VIP groups were not significant. Further, the GH released from the system that perifused the pituitary alone (10(-8)M VIP) was significantly higher than that containing both the hypothalamus and the pituitary fragments (P less than 0.05). The relative lower GH secretory response to the same dose of VIP applied to the hypothalamus-pituitary suggests that the perifused caprine hypothalamus may release an inhibitory factor, such as somatostatin in vitro. These results suggest that VIP stimulates GH release by acting directly on the adenohypophysis of the goat.