Identification of calmodulin isoform-specific binding peptides from a phage-displayed random 22-mer peptide library

Plants express numerous calmodulin (CaM) isoforms that exhibit differential activation or inhibition of CaM-dependent enzymes in vitro; however, their specificities toward target enzyme/protein binding are uncertain. A random peptide library displaying a 22-mer peptide on a bacteriophage surface was constructed to screen peptides that specifically bind to plant CaM isoforms (soybean calmodulin (ScaM)-1 and SCaM-4 were used in this study) in a Ca2+-dependent manner. The deduced amino acid sequence analyses of the respective 80 phage clones that were independently isolated via affinity panning revealed that SCaM isoforms require distinct amino acid sequences for optimal binding. SCaM-1-binding peptides conform to a 1-5-10 ((FILVW)XXX(FILV) XXXX(FILVW)) motif (where X denotes any amino acid), whereas SCaM-4-binding peptide sequences conform to a 1-8-14 ((FILVW)XXXXXX(FAILVW)XXXXX(FILVW)) motif. These motifs are classified based on the positions of conserved hydrophobic residues. To examine their binding properties further, two representative peptides from each of the SCaM isoform-binding sequences were synthesized and analyzed via gel mobility shift assays, Trp fluorescent spectra analyses, and phosphodiesterase competitive inhibition experiments. The results of these studies suggest that SCaM isoforms possess different binding sequences for optimal target interaction, which therefore may provide a molecular basis for CaM isoform-specific function in plants. Furthermore, the isolated peptide sequences may serve not only as useful CaM-binding sequence references but also as potential reagents for studying CaM isoform-specific function in vivo.     

Grassland ecosystems in China: review of current knowledge and research advancement

Grasslands are the dominant landscape in China, accounting for 40% of the national land area. Research concerning China's grassland ecosystems can be chronologically summarized into four periods: (i) pre-1950s, preliminary research and survey of grassland vegetation and plant species by Russians, Japanese and Western Europeans, (ii) 1950-1975, exploration and survey of vegetation, soils and topography as part of natural resource inventory programmes by regional and national institutions mainly led by the Chinese Academy of Sciences, (iii) 1976-1995, establishment of field stations for long-term ecological monitoring and studies of ecosystem processes, (iv) 1996-present, comprehensive studies of community dynamics and ecosystem function integrating multi-scale and multidisciplinary approaches and experimental manipulations.Major findings of scientific significance in China's grassland ecosystem research include: (i) improved knowledge on succession and biogeochemistry of the semi-arid and temperate grassland ecosystems, (ii) elucidation of life-history strategies and diapause characteristics of the native grasshopper species as one of the key grassland pests, and (iii) development of effective management strategies for controlling rodent pests in grassland ecosystems. Opportunities exist for using the natural grasslands in northern China as a model system to test ecosystem theories that so far have proven a challenge to ecologists worldwide.     

Effects of culture conditions on osteogenic differentiation in human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hBMMSCs) must differentiate into osteogenic cells to allow for successful bone regeneration. In this study, we investigated the effects of different combinations of three soluble osteogenic differentiation-inducing factors [L-ascorbic acid (AC), beta-glycerophosphate (betaG), and bone morphogenic protein-2 (BMP-2)] and the presence of a hydroxyapatite (HA) substrate on hBMMSC osteogenic differentiation in vitro. hBMMSCs were cultured in medium containing various combinations of the soluble factors on culture plates with or without HA coating. After 7 days of culture, alkaline phosphatase (ALP) activity, calcium deposition, and osteoprotegerin (OPG) and osteopontin (OPN) expression were measured. The effects of individual and combined factors were evaluated using a factorial analysis method. BMP-2 predominantly affected expression of early markers of osteogenic differentiation (ALP and OPG). HA had the highest positive effect on OPN expression and calcium deposition. The interaction between AC, betaG, and HA had the second highest positive effect on ALP activity.     

MRI Features in a Canine Model of Ischemic Stroke: Correlation between Lesion Volume and Neurobehavioral Status during the Subacute Stage

The purpose of this study was to evaluate the diagnostic value of magnetic resonance imaging (MRI) and assess the correlation between the volume of the ischemic lesion and neurobehavioral status during the subacute stage of ischemic stroke. Ischemic stroke was induced in 6 healthy laboratory beagles through permanent occlusion of the middle cerebral artery (MCAO). T2-weighted and fluid-attenuated inversion recovery (FLAIR) imaging, diffusion-weighted imaging (DWI), measurement of the apparent diffusion coefficient (ADC) ratio, and neurobehavioral evaluation were performed 3 times serially by using a 1.5-T MR system: before and 3 and 10 d after MCAO. Ischemic lesions demonstrated T2 hyperintensity, FLAIR hyperintensity, and DWI hyperintensity. The ADC ratio was decreased initially but then was increased at 10 d after MCAO. Ischemic lesion volumes on T2-weighted and FLAIR imaging were not significantly different from those on DWI. The lesion volume and neurobehavioral score showed strong correlation. Our results suggest that conventional MRI may be a reliable diagnostic tool during the subacute stage of canine ischemic stroke.Abbreviations: ADC, apparent diffusion coefficient; DWI, diffusion-weighted imaging; FLAIR, fluid-attenuated inversion recovery; MCAO, middle cerebral artery occlusion; MRI, magnetic resonance imaging; PWI, perfusion-weighted imagingIn human medicine, stroke is a leading cause of adult mortality and neurologic disability worldwide.¹ Strokes previously were thought to be uncommon in small animals, but the true prevalence is unknown.⁴ These events are now recognized more frequently in dogs because of increased use of magnetic resonance imaging (MRI).^5,14,17Because the infusion of thrombolytic agents, such as urokinase or tissue plasminogen activator, within 3 to 6 h of the onset symptoms is effective in restoring blood flow and improving stroke outcome in humans,¹⁹ the detection of early ischemic changes is now thought to be necessary to improve patient outcome. Computed tomography and conventional MRI are not sufficiently sensitive to predict the presence and extent of ischemic damage during the acute stage after a stroke.^12,20 Therefore several MRI sequences, such as fluid-attenuated inversion recovery (FLAIR), diffusion-weighted imaging (DWI), perfusion-weighted imaging (PWI), and MR angiography, have been developed for early diagnosis and subsequent follow-up of ischemic stroke.³ High-field magnetic strengths (at least 1.5 T) are necessary to perform these sequences.In contrast to the situation in humans, ischemic stroke in many dogs is diagnosed during the subacute stage—24 h to 6 wk after the vascular insult—due to the time lag between the onset of clinical signs to referral and to the lack of standard diagnostic protocols for ischemic stroke in dogs. In most reports of strokes in dogs, the median interval between the onset of neurologic dysfunction and performance of an MRI was more than 2 d.^5,14,17 Whereas DWI has marked sensitivity to very early ischemic changes in the brain, T2-weighted and FLAIR images gradually become more hyperintense later (that is, during the first 24 h after the insult).³ Therefore, hyperintensity on T2-weighted and FLAIR images is believed to be representative of mature lesions.¹⁵ In light of these findings, we hypothesized that conventional MR sequences, such as T2-weighted and FLAIR imaging as well as DWI would be used for the diagnosis of the subacute stage of ischemic stroke in dogs.The purpose of this study was to evaluate the diagnostic value of MRI and assess the correlation between the volume of ischemic lesions and neurobehavioral status during the subacute stage of ischemic stroke in dogs. We therefore investigated the lesion volume of T2-weighted and FLAIR images compared with that on DWI images. Furthermore, we assessed the relationship between the apparent diffusion coefficient (ADC) of the ischemic lesions and the neurobehavioral status of the dogs.     

Essential requirements for robust signaling in Hfq dependent small RNA networks

Bacteria possess networks of small RNAs (sRNAs) that are important for modulating gene expression. At the center of many of these sRNA networks is the Hfq protein. Hfq's role is to quickly match cognate sRNAs and target mRNAs from among a large number of possible combinations and anneal them to form duplexes. Here we show using a kinetic model that Hfq can efficiently and robustly achieve this difficult task by minimizing the sequestration of sRNAs and target mRNAs in Hfq complexes. This sequestration can be reduced by two non-mutually exclusive kinetic mechanisms. The first mechanism involves heterotropic cooperativity (where sRNA and target mRNA binding to Hfq is influenced by other RNAs bound to Hfq); this cooperativity can selectively decrease singly-bound Hfq complexes and ternary complexes with non-cognate sRNA-target mRNA pairs while increasing cognate ternary complexes. The second mechanism relies on frequent RNA dissociation enabling the rapid cycling of sRNAs and target mRNAs among different Hfq complexes; this increases the probability the cognate ternary complex forms before the sRNAs and target mRNAs degrade. We further demonstrate that the performance of sRNAs in isolation is not predictive of their performance within a network. These findings highlight the importance of experimentally characterizing duplex formation in physiologically relevant contexts with multiple RNAs competing for Hfq. The model will provide a valuable framework for guiding and interpreting these experiments.     

Genome-wide analysis of the Chinese sturgeon sox gene family: identification,characterisation and expression profiles of different tissues

The sox family is assumed to be responsible for a number of developmental systems. Genome sequencing technology makes it possible to scan sox genes and conduct characteristic analyses of different species. In fish, full characterisation of sox genes at the genome-wide level has been reported for pufferfish Takifugu rubripes, medaka Oryzias latipes, tilapia Oreochromis niloticus and channel catfish Ictalurus punctatus. However, no systematic investigation of the sox family in sturgeons (Acipenseridae) has been reported to date. This study conducted genome-wide identification of the sox genes in the Chinese sturgeon Acipenser sinensis and profiled their tissue distribution between male and female individuals. In total, 19 sox genes were identified, including soxb1, b2, c, d, e, f and h, in the Chinese sturgeon. Genomic structure analysis indicated relatively conserved exon–intron structures in each sox group and phylogenetic analysis supported the previous classification of the sox family. Most of the sox genes showed a tissue-specific expression pattern, indicating the possible involvement of Chinese sturgeon sox genes at different developmental processes such as cardiac and gonadal development. This study provides a comprehensive resource of Chinese sturgeon sox genes and enables a better understanding of the evolution and function of the sox family.