Cloning and characterization of a ribosomal protein L23a from Haemaphysalis qinghaiensis eggs by immuno screening of a cDNA expression library

A primary cDNA library with a size of 1.34 × 10⁶ PFU was constructed from Haemaphysalis qinghaiensis eggs and was immunoscreened with rabbit anti-H. qinghaiensis serum. One clone (Hq22, named following those clones obtained from adult Haemaphysalis qinghaiensis cDNA library which we constructed before) screened from the cDNA library was selected randomly for sequencing. The entire sequence of the clone was subsequently obtained using rapid amplification of the cDNA ends (RACE). A search of the cloned sequence against GenBank revealed that it related to ribosomal protein L23a (Rpl23a) and had a high percentage similarity to this protein from different species. Conserved domains for Rpl23a were also identified in the cloned sequence. Expression analysis by RT-PCR showed that this gene is expressed in salivary glands, midguts, other tissues and different developmental stages of H. qinghaiensis. Based on the H. qinghaiensis Rpl23a sequence, open reading frames (ORF) of Rpl23a of Heamaphysalis longicornis and Boophilus microplus were also cloned and were performed for comparison with Rpl23a of H. qinghaiensis and other organisms as well. Vaccine based on Rpl23a recombinant protein cannot protect sheep against H. qinghaiensis.     

Calcium-calmodulin is required for abscisic acid-induced antioxidant defense and functions both upstream and downstream of H2O2 production in leaves of maize (Zea mays) plants

* Using pharmacological and biochemical approaches, the role of calmodulin (CaM) and the relationship between CaM and hydrogen peroxide (H(2)O(2)) in abscisic acid (ABA)-induced antioxidant defense in leaves of maize (Zea mays) plants were investigated. * Treatment with ABA or H(2)O(2) led to significant increases in the concentration of cytosolic Ca(2+) in the protoplasts of mesophyll cells and in the expression of the calmodulin 1 (CaM1) gene and the content of CaM in leaves of maize plants, and enhanced the expression of the antioxidant genes superoxide dismutase 4 (SOD4), cytosolic ascorbate peroxidase (cAPX), and glutathione reductase 1 (GR1) and the activities of the chloroplastic and cytosolic antioxidant enzymes. The up-regulation of the antioxidant enzymes was almost completely blocked by pretreatments with two CaM antagonists. * Pretreatments with CaM antagonists almost completely inhibited ABA-induced H(2)O(2) production throughout ABA treatment, but pretreatment with an inhibitor or scavenger of reactive oxygen species (ROS) did not affect the initial increase in the contents of CaM induced by ABA. * Our results suggest that Ca(2+)-CaM is involved in ABA-induced antioxidant defense, and that cross-talk between Ca(2+)-CaM and H(2)O(2) plays a pivotal role in ABA signaling.     

Abscisic acid relations of plants grown on tungsten enriched substrates

The effect of tungsten as a potential inhibitor of ABA biosynthesis in plants was tested both under controlled conditions in the greenhouse or laboratory and in the field at a site naturally enriched with tungsten. Although as predicted in all cases the activity of aldehyde oxidase was strongly inhibited by tungsten, in well watered, greenhouse-grown maize plants increased concentrations of tungsten did not reduce ABA concentration in tissues. Depending on the time of incubation, mostly an accumulation due to an increased net ABA synthesis could be observed. Under drought stress, however, tungsten may have an inhibitory effect on ABA accumulation. Field experiments with drought-stressed clover and dandelion plants from a habitat close to a former tungsten mine confirm those of the greenhouse and laboratory. The ineffectiveness of tungsten in decreasing ABA levels in well-watered plants and its minimal effects in inhibiting ABA accumulation of droughted plants indicate that it is unsuitable as an inhibitor of ABA biosynthesis.     

Secondary metabolites from Commiphora opobalsamum and their antiproliferative effect on human prostate cancer cells

A cycloartane-type triterpenoid (1), an aliphatic alcohol glycoside (2), an eudesmane-type sesquiterpenoid (3), and a guaiane-type sesquiterpenoid (4) were isolated from the resinous exudates of Commiphora opobalsamum along with six known sesquiterpenoids (5-10). Their structures were established by extensive analysis of their 1D and 2D NMR spectroscopic data and chemical methods. The isolated compounds 1-3 and 5-9 were tested against human prostate cancer cell PC 3 and LNCaP. Among them, 1 and 2 showed moderate antiproliferative effects on human prostate cancer cell lines with IC50 values ranging from 5.7 to 23.6 microM; they were also able to inhibit the expression of androgen receptor (AR) in LNCaP cells. The six sesquiterpenoids were inactive in the bioassays.     

Culture and fusion of pollen protoplasts of Brassica oleracea L. var. italica with haploid mesophyll protoplasts of B. rapa L. ssp. pekinensis

Hybrid callus was formed from the successful protoplast fusion between pollen protoplasts of Brassica oleracea var. italica and haploid mesophyll protoplasts of Brassica rapa. The pollen protoplast isolation frequency in broccoli was highly related to the ratio of trinucleate pollens in the male gametophyte population. Large quantities of pollen protoplasts with high vigor could be isolated, and the isolation frequency reached up to 90% in 6.0-7.0 mm long flower buds with about 94.7% trinucleate-stage pollens. Pollen protoplasts could be collected and purified by discontinuous gradient centrifugation. In 1% Na-alginate embedding culture, cell divisions were observed but no further development was found. The haploid mesophyll protoplasts were isolated from in vitro haploid plants of B. rapa. Results strongly showed the variability in culturability of mesophyll protoplasts from different haploid lines. Both pollen protoplasts and haploid mesophyll protoplasts retained a stable round shape in the designed prefusion solution with an osmotic pressure of 0.74 osmol/kg. Polyethylene glycol was used for the protoplast fusion, and 40% polyethylene glycol 4000 enabled the highest fusion frequency of about 20%. Some postfusion protoplasts showed cell divisions up to callus proliferation. Calli were screened by random amplified polymorphic DNA analysis for their hybrid character. Results revealed the existence of the hybrid calli. Some of the hybrid calli grew well with green color and shoot primordia. According to our knowledge, this is the first report about a hybrid formation between two haploid protoplasts. Potential comprehensive applications, as well as problems of this technique, are discussed.     

Directed evolution for increased chitinase activity

Directed evolution through DNA shuffling and screening was used to enhance the catalytic ability of a fungal, Beauveria bassiana, chitinase, Bbchit1. The Bbchit gene was first linked to various prokaryotic signal sequences and expressed in Escherichia coli. The signal peptide, PelB, from Erwinia carotovora resulted in greatest chitinase secretion into broth. The nucleotide sequence expressing PelB signal peptide was then incorporated into an E. coli vector to express Bbchit1 variants generated by three rounds of DNA shuffling. A Bbchit1 library with 150,000 variants was constructed with a nucleotide point mutation frequency of 0.6% and screened for chitinolytic activity. Two Bbchit1 variants (SHU-1 and SHU-2) were selected that showed increased chitinolytic activity compared to the wild type. Sequence analysis of these variants revealed mutations in amino acid residues that would not normally be considered for rational design of improved chitinase activity. The amino acid substitutions occurred outside of the two putative substrate-binding sites and the catalytic region.     

Adiponectin improves endothelial function in hyperlipidemic rats by reducing oxidative/nitrative stress and differential regulation of eNOS/iNOS activity

Plasma adiponectin level is significantly reduced in patients with metabolic syndrome, and vascular dysfunction is an important pathological event in these patients. However, whether adiponectin may protect endothelial cells and attenuate endothelial dysfunction caused by metabolic disorders remains largely unknown. Adult rats were fed with a regular or a high-fat diet for 14 wk. The aorta was isolated, and vascular segments were incubated with vehicle or the globular domain of adiponectin (gAd; 2 mug/ml) for 4 h. The effect of gAd on endothelial function, nitric oxide (NO) and superoxide production, nitrotyrosine formation, gp91(phox) expression, and endothelial nitric oxide synthase (eNOS)/inducible NOS (iNOS) activity/expression was determined. Severe endothelial dysfunction (maximal vasorelaxation in response to ACh: 70.3 +/- 3.3 vs. 95.2 +/- 2.5% in control, P < 0.01) was observed in hyperlipidemic aortic segments, and treatment with gAd significantly improved endothelial function (P < 0.01). Paradoxically, total NO production was significantly increased in hyperlipidemic vessels, and treatment with gAd slightly reduced, rather than increased, total NO production in these vessels. Treatment with gAd reduced (-78%, P < 0.01) superoxide production and peroxynitrite formation in hyperlipidemic vascular segments. Moreover, a moderate attenuation (-30%, P < 0.05) in gp91(phox) and iNOS overexpression in hyperlipidemic vessels was observed after gAd incubation. Treatment with gAd had no effect on eNOS expression but significantly increased eNOS phosphorylation (P < 0.01). Most noticeably, treatment with gAd significantly enhanced eNOS (+83%) but reduced iNOS (-70%, P < 0.01) activity in hyperlipidemic vessels. Collectively, these results demonstrated that adiponectin protects the endothelium against hyperlipidemic injury by multiple mechanisms, including promoting eNOS activity, inhibiting iNOS activity, preserving bioactive NO, and attenuating oxidative/nitrative stress.     

Synergy between docosahexaenoic acid and butyrate elicits p53-independent apoptosis via mitochondrial Ca(2+) accumulation in colonocytes

Butyrate, a short-chain fatty acid fiber fermentation product, induces colonocyte apoptosis in part via a Fas-mediated (extrinsic) pathway. In previous studies, we demonstrated that docosahexaenoic acid (DHA, 22:6(Delta4,7,10,13,16,19)) enhances the effect of butyrate by increasing mitochondrial lipid oxidation and mitochondrial Ca(2+)-dependent apoptosis in the colon. In this study, we further examined the mechanism of DHA-butyrate synergism in 1) human colon tumor (HCT-116 isogenic p53+/+ vs. p53-/-) cells and 2) primary cultures of rat colonic crypts. Herein, we show that DHA and butyrate promote apoptosis by enhancing mitochondrial Ca(2+) accumulation in both isogenic cell lines. Ca(2+) accumulation and apoptosis were inhibited by blockade of mitochondrial uniporter-mediated Ca(2+) uptake. In addition, Mito-Q, a mitochondria-targeted antioxidant, also blocked apoptosis induced by DHA and butyrate. In complementary experiments, rats were fed diets supplemented with either corn oil (control, contains no DHA) or fish oil (contains DHA). Colonic crypts were isolated and incubated with or without butyrate, after which the mitochondria-to-cytosol Ca(2+) ratio and crypt viability were measured. No significant difference (P > 0.05) in basal mitochondrial Ca(2+) levels was observed between fish oil- or corn oil-fed animals. In contrast, when fish oil was the dietary lipid source, crypts incubated with butyrate exhibited a significant increase (3.6-fold, P < 0.001) in mitochondrial Ca(2+) compared with corn oil plus butyrate treatment. On the basis of these data, we propose that the combination of DHA and butyrate compared with butyrate alone further enhances colonocyte apoptosis by inducing a p53-independent, oxidation-sensitive, mitochondrial Ca(2+) -dependent (intrinsic) pathway.     

A New Retroposed Gene in <Emphasis Type="Italic">Drosophila</Emphasis> Heterochromatin Detected by Microarray-Based Comparative Genomic Hybridization

A genomic pattern of new gene origination is often dependent on a genomic method that can efficiently identify a statistically adequate number of recently originated genes. The heterochromatic regions have often been viewed as genomic deserts with low coding potential and thus a low flux of new genes. However, increasing reports revealed unexpected roles of heterochromatic regions in the evolution of genes and genomes. We identified recently retroposed genes that originated in heterochromatic regions in Drosophila, by developing microarray-based comparative genomic hybridization (CGH) with multiple species. This new gene family, named Ifc-2h, originated in the common ancestor of the clade of D. simulans, D. mauritiana, and D. sechellia. The sequence features and phylogenetic distribution indicated that Ifc-2h resulted from the retroposition from its parental gene, Infertile crescent (Ifc), and integrated into heterochromatic region of common ancester of the three sibling species 2 million years ago. Expression analysis revealed that Ifc-2h had developed a new expression pattern by recruiting a putative regulatory element from its target sequence. The distribution of indel variation in Ifc-2h of D. simulans and D. mauritiana revealed a significant sequence constraint, suggesting that the Ifc-2h gene may be functional. These analyses cast fresh insight into the evolution of heterochromatin and the origin of its coding regions. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Martin Kreitman]     

Adenoviruses use lactoferrin as a bridge for CAR-independent binding to and infection of epithelial cells

Most adenoviruses bind to the coxsackie- and adenovirus receptor (CAR). Surprisingly, CAR is not expressed apically on polarized cells and is thus not easily available to viruses. Consequently, alternative mechanisms for entry of coxsackievirus and adenovirus into cells have been suggested. We have found that tear fluid promotes adenovirus infection, and we have identified human lactoferrin (HLf) as the tear fluid component responsible for this effect. HLf alone was found to promote binding of adenovirus to epithelial cells in a dose-dependent manner and also infection of epithelial cells by adenovirus. HLf was also found to promote gene delivery from an adenovirus-based vector. The mechanism takes place at the binding stage and functions independently of CAR. Thus, we have identified a novel binding mechanism whereby adenovirus hijacks HLf, a component of the innate immune system, and uses it as a bridge for attachment to host cells.