Genetic inactivation of NPC1L1 protects against sitosterolemia in mice lacking ABCG5/ABCG8

Mice lacking Niemann-Pick C1-Like 1 (NPC1L1) (NPC1L1(-/-)mice) exhibit a defect in intestinal absorption of cholesterol and phytosterols. However, wild-type (WT) mice do not efficiently absorb and accumulate phytosterols either. Cell-based studies show that NPC1L1 is a much weaker transporter for phytosterols than cholesterol. In this study, we examined the role of NPC1L1 in phytosterol and cholesterol trafficking in mice lacking ATP-binding cassette (ABC) transporters G5 and G8 (G5/G8(-/-) mice). G5/G8(-/-) mice develop sitosterolemia, a genetic disorder characterized by the accumulation of phytosterols in blood and tissues. We found that mice lacking ABCG5/G8 and NPC1L1 [triple knockout (TKO) mice] did not accumulate phytosterols in plasma and the liver. TKO mice, like G5/G8(-/-) mice, still had a defect in hepatobiliary cholesterol secretion, which was consistent with TKO versus NPC1L1(-/-) mice exhibiting a 52% reduction in fecal cholesterol excretion. Because fractional cholesterol absorption was reduced similarly in NPC1L1(-/-) and TKO mice, by subtracting fecal cholesterol excretion in TKO mice from NPC1L1(-/-) mice, we estimated that a 25g NPC1L1(-/-) mouse may secrete about 4 mumol of cholesterol daily via the G5/G8 pathway. In conclusion, NPC1L1 is essential for phytosterols to enter the body in mice.     

The cytosolic protein talin induces an intermediate affinity integrin alphaLbeta2

The integrin alphaLbeta2 mediates leukocyte adhesion and migration that are required for a functional immune system. It is known that inside-out signaling triggers alphaLbeta2 conformational changes, which affect its ligand-binding affinity. At least three alphaLbeta2 affinity states (low, intermediate, and high) were described. The cytosolic protein talin connects alphaLbeta2 to the actin filament. The talin head domain is also known to activate alphaLbeta2 ligand binding. However, it remains to be determined whether talin promotes an intermediate or high affinity alphaLbeta2. In this study using transfectants and T cells, we showed that talin induced an intermediate affinity alphaLbeta2 that adhered constitutively to its ligand intercellular adhesion molecule (ICAM)-1 but not ICAM-3. Adhesion to ICAM-3 was induced when an additional exogenous activating agent was included. Similar profiles were observed with soluble ICAMs. In addition, the intermediate affinity alphaLbeta2 induced by talin allowed adhesion and migration of T cells on immobilized ICAMs.     

A new long terminal repeat (LTR) sequence allows to identify J genome from J<Superscript>S</Superscript> and St genomes of <Emphasis Type="Italic">Thinopyrum intermedium</Emphasis>

A repetitive sequence of 491 bp, named pMD232-500, was isolated from S. cereale cv. Kustro using wheat SSR marker Xgwm232. GenBank BLAST search revealed that the sequence of pMD232-500 was highly similar to a part of retrotransposon Nusif-1. Fluorescence in situ hybridization (FISH) analysis using pMD232-500 as probe indicated that only 14 Thinopyrum intermedium chromosomes and all the chromosomes of S. cereale cv. Kustro bear FISH signals, however, no FISH signals were observed on Dasypyrum villosum chromosomes. In addition, the FISH signals were distributed on whole arms except their terminal regions. Further genomic in situ hybridization (GISH) analysis using genomic DNA from Pseudoroegneria spicata indicated that the 14 Th. intermedium chromosomes bearing FISH signals should belong to J genome. Thereafter, the repetitive elements pMD232-500 showed the unambiguous features of genomic constitution of Th. intermedium. In addition, the results in the present study have indicated the similarity of genomes from Th. intermedium and S. cereale.     

Molecular Population Genetics of Rice Domestication

Domestication is a selection process that genetically modifies species to meet human needs. A most intriguing feature of domestication is the extreme phenotypic diversification among breeds. What could be the ultimate source of such genetic variations? Another notable outcome of artificial selection is the reduction in the fitness of domesticated species when they live in the wild without human assistance. The complete sequences of the two subspecies of rice cultivars provide an opportunity to address these questions. Between the two subspecies, we found much higher rates of non‐synonymous (N) than synonymous (S) substitutions and the N/S ratios are higher between cultivars than between wild species. Most interestingly, substitutions of highly dissimilar amino acids that are deleterious and uncommon between natural species are disproportionately common between the two subspecies of rice. We suggest strong selection in the absence of effective recombination may be the driving force, which we called the domestication‐associated Hill‐Robertson effect. These hitchhiking mutations may contribute to some fitness reduction in cultivars. Comparisons of the two genomes also reveal the existence of highly divergent regions in the genomes. Haplotypes in these regions often form highly polymorphic linkage blocks that are much older than speciation between wild species. Genes from such regions could contribute to the differences between indica and japonica and are likely to be involved in the diversifying selection under domestication. Their existence suggests that the amount of genetic variation within the single progenitor species Oryza rufipogon may be insufficient to account for the variation among rice cultivars, which may come from a more inclusive gene pool comprising most of the A‐genome wild species. Genes from the highly polymorphic regions also provide strong support for the independent domestication of the two subspecies. The genomic variation in rice has revealing implications for studying the genetic basis of indica‐japonica differentiation under rice domestication and subsequent improvement.     

Production of Transgenic Rice Homozygous Lines with Enhanced Resistance to the Rice Brown Planthopper

Mature seed‐derived callus from an elite Chinese japonica rice (Oryza sativa L.) cv. Eyi 105 was cotransformed with two plasmids, pWRG1515 and pRSSGNA1,containing the selectable marker hygromycin phosphotransferase gene (hpt), the reporter β‐glucuronidase gene (gusA) and the snow‐drop (Galanthus nivalis) lectin gene (gna) via particle bombardment. After two rounds of selection on hygromycin‐containing medium, resistant callus was transferred to hygromycin‐containing regeneration medium for plant regeneration. Twenty‐six independent transgenic rice plants were regenerated from 152 bombarded calli with a transformation frequency of 17%. Seventy‐three percent of transgenic plants contained all three genes, which was revealed by PCR/Southern blot analysis. Thirteen out of 19 transgenic plants containing the gna gene expressed GNA (68%) at various levels with the highest expression being approximately 0.5% of total soluble protein. Genetic analysis confirmed Mendelian segregation of transgenes in progeny. From R2 generations with their R1 parentplants showing 3:1 Mendelian segregation patterns, we identified three independent homozygous lines containing and expressing all three transgenes.Insect bioassay and feeding tests showed that these homozygous lines had significant inhibition to the rice brown planthopper (Nilaparvata lugens, BPH) by decreasing BPH survival and overall fecundity, retarding BPH development and reducing BPH feeding.This is the first report that homozygous transgenic rice lines expressing GNA, developed by genetic transformation and through genetic analysis‐based selection, conferred enhanced resistance to BPH, one of the most damaging insect pests in rice.     

Phosphorylation of sucrose synthase at serine 170: occurrence and possible role as a signal for proteolysis

Sequence analysis identified serine 170 (S170) of the maize (Zea mays L.) SUS1 sucrose synthase (SUS) protein as a possible, second phosphorylation site. Maize leaves contained two calcium-dependent protein kinase activities and a calcium-independent kinase activity with characteristics of an sucrose non-fermenting 1 (SNF1)-related protein kinase. Phosphorylation of the novel S170 and the known serine 15 (S15) site by these protein kinases was determined in peptide substrates and detected in SUS1 protein substrates utilizing sequence- and phosphorylation-specific antibodies. We demonstrate phosphorylation of S170 in vitro and in vivo. The calcium-dependent protein kinases phosphorylated both S170 and S15, whereas SNF1-related protein kinase activity was restricted to S15. Calcium-dependent protein-kinase-mediated S170 and S15 phosphorylation kinetics were determined in wild-type and mutant SUS1 substrates. These analyses revealed that kinase specificity for S170 was threefold lower than that for S15, and that phosphorylation of S170 was stimulated by prior phosphorylation at the S15 site. The SUS-binding peptides encoded by early nodulin 40 (ENOD40) specifically antagonized S170 phosphorylation in vitro. A model wherein S170 phosphorylation functions as part of a mechanism targeting SUS for proteasome-mediated degradation is supported by the observations that SUS proteolytic fragments: (i) were detected and possessed relatively high phosphorylated-S170 (pS170) stoichiometry; (ii) were spatially coincident with proteasome activity within developing leaves; and (iii) co-sedimented with proteasome activity. In addition, full-length pS170-SUS protein was less stable than S170-SUS in cultured leaf segments and was stabilized by proteasome inhibition. Post-translational control of SUS protein level through pS170-promoted proteolysis may explain the specific and significant decrease in SUS abundance that accompanies the sink-to-source transition in developing maize leaves.     

Lactose Induction Increases Production of Recombinant Keratinocyte Growth Factor-2 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Recombinant human keratinocyte growth factor-2 (rhKGF-2) has previously been expressed in Escherichia coli using isopropyl-β-d-thiogalactopyranoside (IPTG), a non-metabolizable and expensive compound, as the inducer. In order to determine whether IPTG could be replaced with the cheap and natural lactose to induce rhKGF-2 expression, we examined the expression of rhKGF-2 in flask culture and 30-l fermentation using lactose as the inducer. The optimized fermentation induced with lactose resulted in 1,382 g of cell mass, corresponding to a 84% enhancement in cell mass compared with IPTG induction. While the expression level of rhKGF-2 induced with lactose was comparable to that induced with IPTG, the solubility of target protein was increased by lactose induction than by IPTG induction. The recombinant protein was further purified by cation exchange and heparin-affinity chromatography. 255 milligrams of pure rhKGF-2 was achieved per liter culture by lactose induction, 52% higher than that obtained by IPTG induction. A preliminary biochemical characterization of purified rhKGF-2 was performed by Western blotting and mitogenic activity analysis, and the results demonstrated that the purified lactose-induced rhKGF-2 could react with anti-human KGF-2 antibody and stimulate the proliferation of FGFR2-IIIb-transfected mouse BaF3 cells as IPTG-induced rhKGF-2 could do.     

Novel 4-amino-furo[2,3-d]pyrimidines as Tie-2 and VEGFR2 dual inhibitors

A novel class of furo[2,3-d]pyrimidines has been discovered as potent dual inhibitors of Tie-2 and VEGFR2 receptor tyrosine kinases (TK) and a diarylurea moiety at 5-position shows remarkably enhanced activity against both enzymes. One of the most active compounds, 4-amino-3-(4-((2-fluoro-5-(trifluoromethyl)phenyl)amino-carbonylamino)phenyl)-2-(4-methoxyphenyl)furo[2,3-d]pyrimidine (7k) is <3 nM on both TK receptors and the activity is rationalized based on the X-ray crystal structure.     

中性内肽酶及其抑制剂在心血管疾病过程中的作用

中性内肽酶是一种属于Ⅱ型跨膜蛋白的肽类内切酶,在肽链的氨基端水解疏水氨基酸肽键,灭活心钠素、肾上腺髓质素、血管紧张素、内皮素等心血管活性肽,在高血压病、心力衰竭、动脉粥样硬化和休克等心血管疾病的发病过程中发挥重要作用。