Ribonuclease H-like gene SMALL GRAIN2 regulates grain size in rice through brassinosteroid signaling pathway

Grain size is a key agronomic trait that determines the yield in plants. Regulation of grain size by brassinosteroids (BRs) in rice has been widely reported. However, the relationship between the BR signaling pathway and grain size still requires further study. Here, we isolated a rice mutant, named small grain2 (sg2), which displayed smaller grain and a semi-dwarf phenotype. The decreased grain size was caused by repressed cell expansion in spikelet hulls of the sg2 mutant. Using map-based cloning combined with a MutMap approach, we cloned SG2, which encodes a plant-specific protein with a ribonuclease H-like domain. SG2 is a positive regulator downstream of GLYCOGEN SYNTHASE KINASE2 (GSK2) in response to BR signaling, and its mutation causes insensitivity to exogenous BR treatment. Genetical and biochemical analysis showed that GSK2 interacts with and phosphorylates SG2. We further found that BRs enhance the accumulation of SG2 in the nucleus, and subcellular distribution of SG2 is regulated by GSK2 kinase activity. In addition, Oryza sativa OVATE family protein 19 (OsOFP19), a negative regulator of grain shape, interacts with SG2 and plays an antagonistic role with SG2 in controlling gene expression and grain size. Our results indicated that SG2 is a new component of GSK2-related BR signaling response and regulates grain size by interacting with OsOFP19.     

Functional analysis of protein disulfide isomerases in blood feeding, viability and oocyte development in Haemaphysalis longicornis ticks

Three protein disulfide isomerases from Haemaphysalis longicornis ticks (designated as HlPDI-1, HlPDI-2, and HlPDI-3) were previously identified. In order to further analyze their biological functions, the dsRNA of each HlPDI gene and one dsRNA combination of HlPDI-1/HlPDI-3 were separately injected into female ticks. Reduction of gene and protein expression of HlPDIs by RNA interference (RNAi) was demonstrated by real-time PCR, RT-PCR and Western blot analysis. In single dsRNA-injected groups, HlPDI-1 RNAi impacted tick blood feeding and oviposition, HlPDI-2 RNAi impacted tick viability and HlPDI-3 RNAi had no significant impact by itself. However, the injection of a combination of HlPDI-1/HlPDI-3 dsRNA had synergistic effects on tick viability. Furthermore, the midgut and cuticle were severely damaged in HlPDI-2 dsRNA-injected ticks and HlPDI-1/HlPDI-3 dsRNA-injected ticks, respectively, and disruption of HlPDI genes led to a significant reduction of disulfide bond-containing vitellogenin (Vg) expression in ticks. These results indicate that PDIs from H. longicornis are involved in blood feeding, viability and oocyte development, probably by mediating the formation of disulfide bond-containing proteins of the ticks and the formation of basement membrane and cuticle components such as extracellular matrix (ECM). This is the first report on the functional analysis of PDI family molecules as well as the interactions of PDI and other molecules in blood-feeding arthropods.     

DNA binding properties of the ecdysteroid receptor in the salivary gland of the female ixodid tick, Amblyomma hebraeum

Salivary gland degeneration in the female tick, Amblyomma hebraeum Koch (Acari: Ixodidae) is controlled by an ecdysteroid hormone. In an earlier study (Mao, H., McBlain, W.A., Kaufman, W.R., 1995. Some properties of the ecdysteroid receptor in the salivary gland of the ixodid tick, Amblyomma hebraeum. Gen. Comp. Endocrinol. 99, 340–348), we demonstrated that a protein component of a salivary gland extract binds to ponasterone A (Pon A) with high affinity (Kd1 nM), suggesting a tick ecdysteroid receptor (EcR). In this study, the Pon A binding protein bound to calf thymus DNA; this binding could be dissociated by Drosophila hsp27 EcRE. The binding protein shifted the [³²P]hsp27 EcRE band on a gel mobility shift assay; formation of the complex with hsp27 EcRE required KCl (optimal concentration was approximately 75 mM). A number of physiologically effective ecdysteroids enhanced the binding with the following order of potency: Pon A>Mur A>Mak A>20E>ecdysone, whereas vertebrate steroids (estradiol, cholesterol, corticosterone, progesterone, testosterone) had no such effect. Using monoclonal antibodies against Drosophila EcR and USP, we found that AG10.2 recognized three bands (90.5, 87.3 and 84 kDa for EcR) and AB11 recognized at least two major bands (50.3 and 47.1 kDa for USP) in the salivary gland extract by western blot analysis. In addition, AB11 supershifted the tick EcR-hsp27 EcRE band on a gel mobility shift assay, indicating that the tick EcR heterodimerized with a USP-like protein for DNA binding. Furthermore, selective mutations to the 15-basepair palindrome of hsp27 EcRE at positions −5, +2, or adding a base to the spacer, resulted in considerably reduced affinity to the tick EcR/USP. We thus propose a sequence similarity of EcREs between A. hebraeum and its insect counterpart.     

Molecular characterization and comparative study of 6 salivary gland metalloproteases from the hard tick, Haemaphysalis longicornis

Six genes encoding metalloproteases were identified from the salivary gland of the hard tick, Haemaphysalis longicornis. Comparative analyses have shown the evolutionary distinct and different mRNA expression patterns of each gene during blood feeding. The proteins are synthesized as proenzymes with a prodomain and a metalloprotease/cysteine-rich domain of the reprolysin family. Within the active site, amino acid substitutions were observed. The recombinant Escherichia coli expression of one gene, hlESTMP1, was performed. The immunoblot analysis and indirect fluorescent assay using anti-hlESTMP1 suggested that this protein is mainly expressed in the cytoplasm of the salivary glands and only the mature form of 34 kDa was detectable. The proenzyme expressed by baculovirus was processed into a mature domain, suggesting that proenzyme activation possibly occurs through a pro-protein convertase dependent pathway. The presence of these diverse enzymes might contribute to the greater functional complexity of bioactive molecules in tick saliva to facilitate blood feeding.     

NCL1, a novel gene for a non-essential nuclear protein in Saccharomyces cerevisiae

The nucleolar protein Nop2p is an essential gene product that is required for pre-rRNA processing and ribosome biogenesis in Saccharomyces cerevisiae (Hong, B. et al., 1997, Mol. Cell. Biol., 17, 378–388). A search for proteins similar to Nop2p identified a novel yeast gene product that also shares significant homology with the human proliferation associated nucleolar protein p120. The gene encoding this 78 kDa protein was termed NCL1 (for nuclear protein 1; corresponding to YBL024w). Ncl1p and Nop2p contain an evolutionarily conserved motif that has been termed the ‘NOL1/NOP2/fmu family signature' (NOL1 encodes p120). Epitope tagged Ncl1p was found to be localized to the nucleus, including the nucleolus, and was concentrated at the nuclear periphery. NCL1 is not essential. Strains containing a disruption of NCL1, or strains overexpressing NCL1, grow essentially identically to wildtype NCL1 strains on a number of different media and at different temperatures. Disruption of NCL1 does not affect steady-state levels of large and small ribosome subunits, monoribosomes, and polyribosomes. However, disruption of NCL1 leads to increased sensitivity to the antibiotic paromomycin.     

Effect of the level of dry matter and protein and degradation rate of starch on in vitro ruminal fermentation

Effects of starch type, feeding level and level of a mixed N source upon rumen fermentation were examined using an in vitro rumen simulation system (i.e., SIMCO) with a fluid volume of 1100 ml and liquid dilution rate of approximately 0.07/h. Two sources of starch, two feeding levels and two levels of N were examined in an experiment following a 2 × 2 × 2 factorial arrangement of treatments. The starch sources were raw slowly degrading (R), and cooked fast degrading (C), potato starch and constituted 450 g/kg diet dry matter (DM). The remaining was supplied in the form of a grass hay mixture. The feeding levels were 20 (DM20) and 40 (DM40) g DM/d and the diet N level was either low (N1) or high (N2), using peptone and ammonia salt additions in the buffer. Two simulations of 10 days each were completed. The amount of bicarbonate in buffer was varied according to feeding level, and pH (average 6.3) did not differ between treatments. An increased degradation rate of starch (R vs. C) depressed neutral detergent fibre (aNDFom) digestibility (0.46 vs. 0.36) and organic matter true digestibility (OMTD; 0.73 vs. 0.68), but there were no other treatment effects on digestibility. The VFA production efficiency (average 0.47 g VFA/g OMTD) was not affected by the main treatments, although an interaction between starch type and feeding level occurred. A decline in protozoa rating over the 10 day simulations was more apparent at the lower feeding level (DM20), indicating poor growth conditions for protozoa. Higher microbial efficiency (11.0 vs. 8.9 mg microbial N/g OMTD) and a higher proportion of propionate (272 vs. 207 mmol/mol VFA) occurred at DM20 compared to DM40. Increased degradation rate of starch (i.e., R vs. C) resulted in an improved microbial N efficiency (8.8 vs. 11.2 mg/g OMTD) and an increase in the proportion of propionate (226 vs. 253 mmol/mol VFA) at the expense of acetate (610 vs. 591 mmol/mol VFA). Increasing the level of N (i.e., N1 vs. N2) improved the microbial N efficiency (9.2 vs. 10.7 mg/g OMTD) and increased the proportion of propionate (230 vs. 249 mmol/mol VFA) and butyrate (100 vs. 119 mmol/mol VFA) at the expense of acetate (629 vs. 572 mmol/mol VFA). Interactions between starch type and N level upon VFA patterns were apparent. Results support earlier findings in that carbohydrate degradation rate is an important factor determining microbial growth rates and VFA distribution.     

Proteomic Analysis of Cattle Tick Rhipicephalus (Boophilus) microplus Saliva: A Comparison between Partially and Fully Engorged Females

The cattle tick Rhipicephalus (Boophilus) microplus is one of the most harmful parasites affecting bovines. Similarly to other hematophagous ectoparasites, R. microplus saliva contains a collection of bioactive compounds that inhibit host defenses against tick feeding activity. Thus, the study of tick salivary components offers opportunities for the development of immunological based tick control methods and medicinal applications. So far, only a few proteins have been identified in cattle tick saliva. The aim of this work was to identify proteins present in R. microplus female tick saliva at different feeding stages. Proteomic analysis of R. microplus saliva allowed identifying peptides corresponding to 187 and 68 tick and bovine proteins, respectively. Our data confirm that (i) R. microplus saliva is complex, and (ii) that there are remarkable differences in saliva composition between partially engorged and fully engorged female ticks. R. microplus saliva is rich mainly in (i) hemelipoproteins and other transporter proteins, (ii) secreted cross-tick species conserved proteins, (iii) lipocalins, (iv) peptidase inhibitors, (v) antimicrobial peptides, (vii) glycine-rich proteins, (viii) housekeeping proteins and (ix) host proteins. This investigation represents the first proteomic study about R. microplus saliva, and reports the most comprehensive Ixodidae tick saliva proteome published to date. Our results improve the understanding of tick salivary modulators of host defense to tick feeding, and provide novel information on the tick-host relationship.     

Hemalin, a thrombin inhibitor isolated from a midgut cDNA library from the hard tick Haemaphysalis longicornis

A full-length sequence of a thrombin inhibitor (designated as hemalin) from the midgut of parthenogenetic Haemaphysalis longicornis has been identified. Sequence analysis shows that this gene belongs to the Kunitz-type family, containing two Kunitz domains with high homology to boophilin, the thrombin inhibitor from Rhipicephalus (Boophilus) microplus. The recombinant protein expressed in insect cells delayed bovine plasma clotting time and inhibited both thrombin-induced fibrinogen clotting and platelet aggregation. A 20-kDa protein was detected from the midgut lysate with antiserum against recombinant hemalin. The gene is expressed at all stages of the tick except for the egg stage, and hemalin mRNA mainly in the midgut of the female adult tick. Real-time PCR analysis shows that this gene has a distinctly high expression level in the rapid bloodsucking period of the larvae, nymphs, and adults. Disruption of the hemalin gene by RNA interference led to a 2-day extension of the tick blood feeding period, and 27.7% of the RNA-treated ticks did not successfully complete the blood feeding. These findings indicate that the newly identified thrombin inhibitor from the midgut of H. longicornis might play an important role in tick blood feeding.     

Isolation and characterization of a metagenome-derived and cold-active lipase with high stereospecificity for (R)-ibuprofen esters

We report on the isolation and biochemical characterization of a novel, cold-active and metagenome-derived lipase with a high stereo-selectivity for pharmaceutically important substrates. The respective gene was isolated from a cosmid library derived from oil contaminated soil and designated lipCE. The deduced aa sequence indicates that the protein belongs to the lipase family l.3, with high similarity to Pseudomonas fluorescens lipases containing a C-terminal secretion signal for ABC dependent transport together with possible motifs for Ca²⁺-binding sites. The overexpressed protein revealed a molecular weight of 53.2 kDa and was purified by refolding from inclusion bodies after expression in Escherichia coli. The optimum temperature of LipCE was determined to be 30 °C. However, the enzyme still displayed 28% residual activity at 0 °C and 16% at −5 °C. Calcium ions strongly increased activity and thermal stability of the protein. Further detailed biochemical characterization of the recombinant enzyme showed an optimum pH of 7 and that it retained activity in the presence of a range of metal ions and solvents. A detailed analysis of the enzyme's substrate spectrum with more than 34 different substrates indicated that the enzyme was able to hydrolyze a wide variety of substrates including the conversion of long chain fatty acid substrates with maximum activity for pNP-caprate (C₁₀). Furthermore LipCE was able to hydrolyze stereo-selectively ibuprofen-pNP ester with a high preference for the (R) enantiomer of >91% ee and it demonstrated selectivity for esters of primary alcohols, whereas esters of secondary or tertiary alcohols were nearly not converted.     

A Proteomics Approach to Characterizing Tick Salivary Secretions

The saliva of ticks contains a complex mixture of bioactive molecules including proteins that modulate host responses ensuring successful feeding. The limited amount of saliva that can be obtained from ticks has hampered characterization of salivary proteins using traditional protein chemistry. Recent improvements in two-dimensional gel electrophoresis, mass spectrometry, and bioinformatics provide new tools to characterize small amounts of protein. These methods were employed to characterize salivary proteins from Amblyomma americanum and Amblyomma maculatum. Salivation was induced by injection of dopamine and theophylline. It was necessary to desalt and concentrate saliva before analysis by 2-D electrophoresis. Comparison of 1-D and 2-D gel patterns revealed that the major protein component of saliva did not appear on 2-D gels. Characterization of this protein showed that it was identical to the major protein present in the hemolymph of both tick species. Protein profiles obtained by 1-D and 2-D gel electrophoresis were similar for both tick species, however, higher concentrations of lower molecular weight proteins were present in A. maculatum. Protein analysis by MALDI-TOF mass spectrometry and western blot analysis showed that except for the most abundant protein with a molecular weight of 95 kDa, all of the proteins detected were of host origin. It is not known if this is an artifact of the collection method or has physiological significance. In either case, in these species of ticks, host proteins will have to be removed from saliva samples prior to 2-D analysis in order to characterize lower abundance proteins of tick origin. This revised version was published online in July 2006 with corrections to the Cover Date.     

A Proteomics Approach to Characterizing Tick Salivary Secretions

The saliva of ticks contains a complex mixture of bioactive molecules including proteins that modulate host responses ensuring successful feeding. The limited amount of saliva that can be obtained from ticks has hampered characterization of salivary proteins using traditional protein chemistry. Recent improvements in two-dimensional gel electrophoresis, mass spectrometry, and bioinformatics provide new tools to characterize small amounts of protein. These methods were employed to characterize salivary proteins from Amblyomma americanum and Amblyvomma maculatum. Salivation was induced by injection of dopamine and theophylline. It was necessary to desalt and concentrate saliva before analysis by 2-D electrophoresis. Comparison of 1-D and 2-D gel patterns revealed that the major protein component of saliva did not appear on 2-D gels. Characterization of this protein showed that it was identical to the major protein present in the hemolymph of both tick species. Protein profiles obtained by 1-D and 2-D gel electrophoresis were similar for both tick species, however, higher concentrations of lower molecular weight proteins were present in A. maculatum. Protein analysis by MALDI-TOF mass spectrometry and western blot analysis showed that except for the most abundant protein with a molecular weight of 95 kDa, all of the proteins detected were of host origin. It is not known if this is an artifact of the collection method or has physiological significance. In either case, in these species of ticks, host proteins will have to be removed from saliva samples prior to 2-D analysis in order to characterize lower abundance proteins of tick origin.     

Purification, characterization and functional analysis of asparagines synthetase encoding by ste10 gene in Ebosin biosynthesis of Streptomyces sp. 139

Ebosin produced by Streptomyces sp. 139 is a novel exopolysaccharide (EPS) with medicinal activity. This paper describes the functional study of ste10, a putative Ebosin biosynthesis gene. ste10 was cloned and expressed in Escherichia coli BL21 and the purified recombinant protein characterized. Ste10 was shown to be able of catalyzing the transfer of amide nitrogen of glutamine to the side chain of aspartate to produce asparagine. Its Km, optimum temperature and pH were determined to be 0.9 mM, 37 °C and 7.38, respectively. After ste10 gene knock-out, the monosaccharide composition of EPS-m produced by the mutant Streptomyces sp. 139 (ste10⁻) was found changed in comparison with that of Ebosin while its antagonist activity for IL-1R decreased significantly. Based on these results, it is concluded that ste10 codes for an asparagine synthetase which may function as a modificator gene of Ebosin during its biosynthesis.     

Effect of sun ginseng methanol extract on lipopolysaccharide-induced liver injury in rats

Sun ginseng (SG) is heat-processed Panax ginseng C.A. Meyer steamed at 120 °C, which has ginsenoside-Rg₃, -Rk₁, and -Rg₅ as its main ginsenoside components. The effect of SG on lipopolysaccharide (LPS)-induced liver injury in rats was investigated in this study. Intravenous injection of LPS induced excessive nitric oxide (^·NO) generation in serum and increased the hepatic mitochondrial thiobarbituric acid-reactive substance (TBA-RS) level. However, the elevated TBA-RS level was significantly lowered by 15 consecutive days of SG administrations. In addition, up-regulated hepatic inducible nitric oxide synthase and heme oxygenase 1 levels in LPS-treated control rats were significantly lowered and increased, respectively, by 100 mg/kg body weight/day of SG administration. These antioxidant effects were thought to be partially related to the deactivation of nuclear factor-κB by SG administration.