In vitro selected peptides bind with thymidylate synthase mRNA and inhibit its translation

Thymidylate synthase (TS), an essential enzyme for catalyzing the biosynthesis of thymidylate, is a critical therapeutic target in cancer therapy. Recent studies have shown that TS functions as an RNA-binding protein by interacting with two different sequences on its own mRNA, thus, repressing translational efficiency. In this study, peptides binding TS RNA with high affinity were isolated using mRNA display from a large peptide library (＞1013 different sequences). The randomized library was subjected up to twelve rounds of in vitro selection and amplification. Comparing the amino acid composition of the selected peptides (12th round, R12) with those from the initial random library (round zero, R0), the basic and aromatic residues in the selected peptides were enriched significantly, suggesting that these peptide regions might be important in the peptide-TS mRNA interaction. Categorizing the amino acids at each random position based on their physicochemical properties and comparing the distributions with those of the initial random pool, an obvious basic charge characteristic was found at positions 1, 12, 17 and 18, suggesting that basic side chains participate in RNA binding. Secondary structure prediction showed that the selected peptides of R12 pool represented a helical propensity compared with R0 pool, and the regions were rich in basic residues. The electrophoretic gel mobility shift and in vitro translation assays showed that the peptides selected using mRNA display could bind TS RNA specifically and inhibit the translation of TS mRNA. Our results suggested that the identified peptides could be used as new TS inhibitors and developed to a novel class of anticancer agents.     

Effect of nano-TiO2 on photochemical reaction of chloroplasts of spinach

The effects of nano-TiO₂ (rutile) on the photochemical reaction of chloroplasts of spinach were studied. The results showed that when spinach was treated with 0.25% nano-TiO₂, the Hill reaction, such as the reduction rate of FeCy, and the rate of evolution oxygen of chloroplasts was accelerated and noncyclic photophosphorylation (nc-PSP) activity of chloroplasts was higher than cyclic photophosphorylation (c-PSP) activity, the chloroplast coupling was improved and activities of Mg²⁺-ATPase and chloroplast coupling factor I (CF₁)-ATPase on the thylakoid membranes were obviously activated. It suggested that photosynthesis promoted by nano-TiO₂ might be related to activation of photochemical reaction of chloroplasts of spinach.     

Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata

Of 49 microbial strains screened for their capabilities to transform ginsenoside Rb₁, Rhizopus stolonifer and Curvularia lunata produced four key metabolites: 3-O-[-d-glucopyranosyl-(1,2)--d-glucopyranosyl]- 20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ene (1), 3-O-[-d-glucopyranosyl-(1,2)--d- glucopyranosyl]-20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ol (2), 3-O-[-d-gluco- pyranosyl-(1,2)--d-glucopyranosyl]-3, 12, 20(S)-trihydroxydammar-24-ene (3), and 3-O--d-glucopyranosyl-3, 12, 20(S)-trihydroxydammar-24-ene (4), identified by TOF-MS, ¹H- and ¹³C-NMR spectral data. Metabolites 1, 3 and 4 were from the incubation with R. stolonifer, and 1 and 2 from the incubation with C. lunata. Compound 2 was identified as a new compound.     

Plant regeneration via organogenesis from adventitious bud explants of a medicinal herb species, <Emphasis Type="Italic">Polygonatum cyrtonema</Emphasis>

Summary Explants derived from adventitious buds, rhizomes, stems, and leaves of a medicinal plant, Polygonatum cyrtonema, were studied for plantlet regeneration, and only adventitious bud explants were able to be regenerated into plantlets. Regeneration was also accompanied by the formation of rhizome-like tissue, the medicinal portion of the plant. The optimum hormone combination for plantlet regenertion was 4.44 μM benzyladenine plus 2.26 μM 2,4-dichlorophenoxyacetic acid, at which new adventitious buds were obtained from 96.6% of the adventitious bud explants, with an average of 5.2 buds per explant. The best medium for root induction was half-strength Murashige and Skoog medium with 4.57 μM α-naphthaleneacetic acid, as 92% of regenerated buds rooted. Regenerated plantlets were successfully transferred to a greenhouse with 86% survival. Histological observation indicated that new adventitious buds originated from the superficial meristematic cell cluster of the granular callus induced from adventitious bud explants via organogenesis.     

Quantitative trait locus analysis of drought tolerance and yield in Maize in China

Drought accounts for significant yield losses in crops. Maize (Zea mays L.) is particularly sensitive to water stress at reproductive stages, and breeding to improve drought tolerance has been a challenge. By use of a linkage map with 121 single sequence repeat (SSR) markers, quantitative trait loci (QTLs) for grain yield and yield components were characterized in the population of the cross X178×B73 under water-stressed and well-watered conditions. Under the well-watered regime, 2, 4, 4, 1, 2, 2, and 3 QTLs were identified for grain yield, 100-kernel weight, kernel number per ear, cob weight per ear, kernel weight per ear, ear weight, and ear number per plant, respectively, whereas under the water-stressed conditions, 1, 5, 2, 6, 1, 3, and 2 QTLs, respectively, were found. The significant phenotypic correlations among yield and yield components to some extent were observed under both water conditions, and some overlaps between the corresponding QTLs were also found. QTLs for grain yield and kernel weight per ear under well-watered conditions and ear weight under both well-watered and water-stressed conditions over-lapped, and all were located on chromosome 1.03 near marker bnlg176. Two other noticeable QTL regions were on chromosome 9.05 and 9.07 near markers umc1657 and bnlg1525; the first corresponded to grain yield, kernel weight per ear, and ear weight under well-watered conditions and kernel number per ear under both water conditions, and the second to grain yield and cob weight per ear under water-stressed conditions and ear number per plant under both water conditions. A comparative analysis of the QTLs herein identified with those described in previous studies for yield and yield components in different maize populations revealed a number of QTLs in common. These QTLs have potential use in molecular marker-assisted selection.     

Roles of Fragile X Mental Retardation Protein in Dopaminergic Stimulation-induced Synapse-associated Protein Synthesis and Subsequent α-Amino-3-hydroxyl-5-methyl-4-isoxazole-4-propionate (AMPA) Receptor Internalization

Fragile X syndrome, the most common form of inherited mental retardation, is caused by the absence of the RNA-binding protein fragile X mental retardation protein (FMRP). FMRP regulates local protein synthesis in dendritic spines. Dopamine (DA) is involved in the modulation of synaptic plasticity. Activation of DA receptors can regulate higher brain functions in a protein synthesis-dependent manner. Our recent study has shown that FMRP acts as a key messenger for DA modulation in forebrain neurons. Here, we demonstrate that FMRP is critical for DA D1 receptor-mediated synthesis of synapse-associated protein 90/PSD-95-associated protein 3 (SAPAP3) in the prefrontal cortex (PFC). DA D1 receptor stimulation induced dynamic changes of FMRP phosphorylation. The changes in FMRP phosphorylation temporally correspond with the expression of SAPAP3 after D1 receptor stimulation. Protein phosphatase 2A, ribosomal protein S6 kinase, and mammalian target of rapamycin are the key signaling molecules for FMRP linking DA D1 receptors to SAPAP3. Knockdown of SAPAP3 did not affect surface expression of α-amino-3-hydroxyl-5-methyl-4-isoxazole-4-propionate (AMPA) GluR1 receptors induced by D1 receptor activation but impaired their subsequent internalization in cultured PFC neurons; the subsequent internalization of GluR1 was also impaired in Fmr1 knock-out PFC neurons, suggesting that FMRP may be involved in subsequent internalization of GluR1 through regulating the abundance of SAPAP3 after DA D1 receptor stimulation. Our study thus provides further insights into FMRP involvement in DA modulation and may help to reveal the molecular mechanisms underlying impaired learning and memory in fragile X syndrome.     

Non-invasive quantification of peripheral arterial volume distensibility and its non-linear relationship with arterial pressure

Arterial wall function is associated with different physiological and clinical factors. Changes in arterial pressure cause major changes in the arterial wall. This study presents a simple non-invasive method to quantify arterial volume distensibility changes with different arterial pressures.The electrocardiogram, finger and ear photoplethysmogram were recorded from 15 subjects with the right arm at five different positions (90°, 45°, 0°, ?45° and ?90° referred to the horizontal level). Arm pulse propagation time was determined by subtracting ear pulse transit time from finger pulse transit time, and was used to obtain arterial volume distensibility. The mean arterial blood pressure with the arm at the horizontal level was acquired, and changes with position were calculated using the hydrostatic principle that blood pressure in the arm is linearly related to its vertical distance from the horizontal level.The mean arm pulse propagation times for the five different positions were 88, 72, 57, 54 and 52 ms, with the corresponding mean arterial volume distensibility of 0.234%, 0.158%, 0.099%, 0.088% and 0.083% per mmHg. For all consecutive changes in arm position, arm pulse propagation time and arterial volume distensibility, were significantly different (all probability P<0.05). The slopes of arm pulse propagation time and arterial volume distensibility against arterial pressure decreased significantly between each consecutive arm position from 90° to ?45° (all P<0.01), indicating significant non-linearity.The experimental results fitted the physiological exponential model and Langewouters’ arctangent model well, and were also comparable to published data with arterial volume distensibility approximately tripling for transmural pressure changes from 101 to 58 mmHg.In conclusion, the inverse and non-linear relationship between arterial volume distensibility and arterial pressure has been quantified using a simple arm positioning procedure, with the greatest effect at low pressures. This work is an important step in developing a simple non-invasive technique for assessing peripheral arterial volume distensibility.     

Substrate specificity and kinetic isotope effect analysis of the Eschericia coli ketopantoate reductase

Ketopantoate reductase (EC 1.1.1.169), an enzyme in the pantothenate biosynthetic pathway, catalyzes the NADPH-dependent reduction of alpha-ketopantoate to form D-(-)-pantoate. The enzyme exhibits high specificity for ketopantoate, with V and V/K for ketopantoate being 5- and 365-fold higher than those values for alpha-ketoisovalerate and 20- and 648-fold higher than those values for alpha-keto-beta-methyl-n-valerate, respectively. For pyridine nucleotides, V/K for beta-NADPH is 3-500-fold higher than that for other nucleotide substrates. The magnitude of the primary deuterium kinetic isotope effects on V and V/K varied substantially when different ketoacid and pyridine nucleotide substrates were used. The small primary deuterium kinetic isotope effects observed using NADPH and NHDPH suggest that the chemical step is not rate-limiting, while larger primary deuterium isotope effects were observed for poor ketoacid and pyridine nucleotide substrates, indicating that the chemical reaction has become partially or completely rate-limiting. The pH dependence of (D)V using ketopantoate was observed to vary from a value of 1.1 at low pH to a value of 2.5 at high pH, while the magnitude of (D)V/K(NADPH) and (D)V/K(KP) were pH-independent. The value of (D)V is large and pH-independent when alpha-keto-beta-methyl-n-valerate was used as the ketoacid substrate. Solvent kinetic isotope effects of 2.2 and 1.2 on V and V/K, respectively, were observed with alpha-keto-beta-methyl-n-valerate. Rapid reaction analysis of NADPH oxidation using ketopantoate showed no "burst" phase, suggesting that product-release steps are not rate-limiting and the cause of the small observed kinetic isotope effects with this substrate pair. Large primary deuterium isotope effects on V and V/K using 3-APADPH in steady-state experiments, equivalent to the isotope effect observed in single turnover studies, suggests that chemistry is rate-limiting for this poorer reductant. These results are discussed in terms of a kinetic and chemical mechanism for the enzyme.     

Global analysis of Arabidopsis/downy mildew interactions reveals prevalence of incomplete resistance and rapid evolution of pathogen recognition

Interactions between Arabidopsis thaliana and its native obligate oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) represent a model system to study evolution of natural variation in a host/pathogen interaction. Both Arabidopsis and Hpa genomes are sequenced and collections of different sub-species are available. We analyzed ～400 interactions between different Arabidopsis accessions and five strains of Hpa. We examined the pathogen's overall ability to reproduce on a given host, and performed detailed cytological staining to assay for pathogen growth and hypersensitive cell death response in the host. We demonstrate that intermediate levels of resistance are prevalent among Arabidopsis populations and correlate strongly with host developmental stage. In addition to looking at plant responses to challenge by whole pathogen inoculations, we investigated the Arabidopsis resistance attributed to recognition of the individual Hpa effectors, ATR1 and ATR13. Our results suggest that recognition of these effectors is evolutionarily dynamic and does not form a single clade in overall Arabidopsis phylogeny for either effector. Furthermore, we show that the ultimate outcome of the interactions can be modified by the pathogen, despite a defined gene-for-gene resistance in the host. These data indicate that the outcome of disease and disease resistance depends on genome-for-genome interactions between the host and its pathogen, rather than single gene pairs as thought previously.