Regulation of flowering time via miR172-mediated APETALA2-like expression in ornamental gloxinia (Sinningia speciosa) / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

We investigated the microRNA172 (miR172)-mediated regulatory network for the perception of changes in external and endogenous signals to identify a universally applicable floral regulation system in ornamental plants, manipulation of which could be economically beneficial. Transgenic gloxinia plants, in which miR172 was either overexpressed or suppressed, were generated using Agrobacterium-mediated transformation. They were used to study the effect of altering the expression of this miRNA on time of flowering and to identify its mRNA target. Early or late flowering was observed in transgenic plants in which miR172 was overexpressed or suppressed, respectively. A full-length complementary DNA (cDNA) of gloxinia (Sinningia speciosa) APETALA2-like (SsAP2-like) was identified as a target of miR172. The altered expression levels of miR172 caused up- or down-regulation of SsAP2-like during flower development, which affected the time of flowering. Quantitative real-time reverse transcription PCR analysis of different gloxinia tissues revealed that the accumulation of SsAP2-like was negatively correlated with the expression of miR172a, whereas the expression pattern of miR172a was negatively correlated with that of miR156a. Our results suggest that transgenic manipulation of miR172 could be used as a universal strategy for regulating time of flowering in ornamental plants.

Effect of Fetal Hypothyroidism on Cardiac Myosin Heavy Chain Expression in Male Rats / O Efeito de Hipotireoidismo Fetal na Expressão da Miosina Cardíaca de Cadeia Pesada em Ratos Macho

Abstract Background: Thyroid hormone deficiency during fetal life could affect the cardiac function in later life. The mechanism underlying this action in fetal hypothyroidism (FH) in rats has not been elucidated thus far. Objective: The aim of this study is to evaluation the effect of FH on cardiac function in male rats and to determine the contribution of α-myosin heavy chain (MHC) and β-MHC isoforms. Methods: Six pregnant female rats were randomly divided into two groups: The hypothyroid group received water containing 6-propyl-2-thiouracil during gestation and the controls consumed tap water. The offspring of the rats were tested in adulthood. Hearts from the FH and control rats were isolated and perfused with langendroff setup for measuring hemodynamic parameters; also, the heart mRNA expressions of α- MHC and β-MHC were measured by qPCR. Results: Baseline LVDP (74.0 ± 3.1 vs. 92.5 ± 3.2 mmHg, p < 0.05) and heart rate (217 ± 11 vs. 273 ± 6 beat/min, p < 0.05) were lower in the FH rats than controls. Also, these results showed the same significance in ±dp/dt. In the FH rats, β-MHC expression was higher (201%) and α- MHC expression was lower (47%) than control. Conclusion: Thyroid hormone deficiency during fetal life could attenuate normal cardiac functions in adult rats, an effect at least in part due to the increased expression of β-MHC to α- MHC ratio in the heart.

Resumo Fundamento: Deficiência de hormônio da tireoide durante vida fetal pode afetar a função cardíaca no futuro. O mecanismo subjacente dessa ação em hipotireoidismo fetal (HF) em ratos ainda não tem explicação. Objetivo: O objetivo desse estudo é avaliar o efeito de HF na função cardíaca em ratos macho e determinar a contribuição da α-miosina de cadeia pesada (α-MCP) e de isoformas β-MCP. Métodos: Seis ratos fêmea gestantes foram aleatoriamente divididas em dois grupos. O grupo do hipotireoidismo recebeu água contendo 6-propil-2-tiouracil durante a gestação, e os ratos no grupo de controle receberam água de torneira. Os filhotes dos ratos foram testados quando atingiram idade adulta. O coração dos ratos HF e controle foram isolados e submetidos a perfusão pelo método de Langendorff para medição de parâmetros hemodinâmicos. Também foram medidas as expressões de mRNA do coração de α-MCP e β-MCP por qPCR. Resultados: PVED de base (74,0 ± 3,1 vs. 92,5 ± 3,2 mmHg, p < 0,05) e pressão arterial (217 ± 11 vs. 273 ± 6 batidas/min, p < 0,05) mostraram-se mais baixas em ratos HF do que em ratos controle. Além disso, esses resultados mostraram a mesma significância em ±dp/dt. Em ratos HF, a expressão de β-MCP foi mais alta (201%) e a de α-MCP foi mais baixa (47%) do que em ratos controle. Conclusão: Deficiência de hormônio da tireoide durante a vida fetal pode enfraquecer funções cardíacas normais em ratos adultos, efeito devido em parte à expressão aumentada de β-MCP em relação a α-MCP no coração.

Characterization of differentially expressed stress-associated proteins in starch granule development under heat stress in wheat (Triticum aestivum L.).

Abiotic stress causes abrupt increase in the expression of stress-associated proteins, which provide tolerance by modulating the defense mechanism of plants. Small heat shock proteins (sHSPs) and anti-oxidant enzymes are important for environmental stress tolerance of the plants. In this study, two full-length cDNAs encoding small heat shock protein (sHSP) and superoxide dismutase (SOD), designated as TasHSP and SODI were identified and characterized from C-306 (thermotolerant) and PBW343 (thermosusceptible) cultivars of wheat (Triticum aestivum L.). An alpha crystalline domain was observed in TasHSP and manganese/iron binding domain in case of SODI. Quantitative real-time PCR showed very high transcript level of TasHSP and SOD in C-306 compared to PBW343 at different stages of growth and against differential heat stress (HS). Under differential HS at milky-dough stage, the fold change in transcript of both TasHSP and SOD was observed maximum in C-306, compared to PBW343. Protein profiling and isoenzymes analysis showed the expression of several heat-stable proteins and prominent isoenzymes of SOD in C-306, compared to PBW343. Scanning electron microscopy (SEM) of starch granules showed globular, well-shaped and more numbers of endospermic cells in C-306, compared to defragmented, irregular shaped and shrunken granules in case of PBW343 under HS treatment (42°C for 2 h). Diurnal change in soluble starch synthase (SSS) activity showed an increase in the activity during afternoon (35°C), compared to morning (29°C) and evening (32°C) in both the cultivars. Under heat stress (42°C for 2 h), a drastic decrease in the SSS activity was observed, due to the thermal denaturation of the enzyme. Thermotolerance capacity analyzed using cell membrane stability (CMS) showed significantly higher CMS in case of C-306, compared to PBW343 at different stages of growth. Findings suggest that abundance of TasHSP and SODI during milky-dough stage plays a very important role in starch granule biosynthesis. The mechanism may be further exploited to develop tolerant wheat cultivar with high quality seeds.

Antibodies to Interferon beta in Patients with Multiple Sclerosis Receiving CinnoVex, Rebif, and Betaferon

Treatment with interferon beta (IFN-beta) induces the production of binding antibodies (BAbs) and neutralizing antibodies (NAbs) in patients with multiple sclerosis (MS). NAbs against IFN-beta are associated with a loss of IFN-beta bioactivity and decreased clinical efficacy of the drug. The objective of this study was to evaluate the incidence and the prevalence of binding antibodies (BAbs) and neutralizing antibodies (NAbs) to IFN-beta in MS patients receiving CinnoVex, Rebif, or Betaferon. The presence of BAbs was studied in serum samples from 124 MS patients using one of these IFN-beta medications by ELISA. The NAbs against IFN-beta were measured in BAb-positive MS patients receiving IFN-beta using an MxA gene expression assay (real-time RT-PCR). Of the 124 patients, 36 (29.03%) had BAbs after at least 12 months of IFN-beta treatment. The proportion of BAb+ was 38.1% for Betaferon, 21.9% for Rebif, and 26.8% for CinnoVex. Five BAb-positive MS patients were lost to follow-up; thus 31 BAb-positive MS patients were studied for NAbs. NAbs were present in 25 (80.6%) of BAb-positive MS patients receiving IFN-beta. In conclusion, the three IFN-beta preparations have different degrees of immunogenicity.

Structurally conserved C-RFa revealed prolactin releasing activity in vitro and gene expression changes in pituitary of seasonally acclimatized carp

Here we show the cloning and characterization of a novel homolog of prepro C-RFa cDNA from Cyprinus carpió. The deduced preprohormone precursor of 115 amino acids leads to a mature bioactive peptide of 20 amino acids with identical sequence to other teleost C-RFa. Modeling of the mature C-RFa peptide highlighted significant similarity to homologous human PrRP20, specifically the conserved amphipathic system defined by the C-terminal alpha-helix. Clearly, the synthetic C-RFa peptide stimulated prolactin release from primary cultured fish pituitary cells. For the first time, significant variation was shown in C-RFa mRNA and protein levels in the hypothalamus and pituitary between summer- and winter-acclimatized carp. Furthermore, C-RFa protein distribution in carp central nervous tissue was visualized by immunodetection in fibers and cells in hypothalamus, olfactory tract, cerebellum and pituitary stalk. In conclusion, we demonstrated the structure conservation of C-RFa in teleosts and mammals and immunopositive cells and fibers for C-RFa in brain areas. Finally, the increase of C-RFa expression suggests the participation of this hypothalamic factor in the mechanism of modulation in PRL expression in carp.

Single-stranded megaprimer splicing through OE-PCR: Construction of full-length Aspergillus niger arginase cDNA.

A useful variant of PCR technique was devised to generate full-length Aspergillus niger arginase cDNA for expression. Briefly, a 450 bp amplicon was first constructed through overlap extension PCR (OE-PCR) by splicing in a 101 nucleotide long single-stranded megaprimer, facilitated by inclusion of an additional, shorter forward primer in the reaction. The amplicon was suitably cloned into pBlueScript to obtain pArg440 and the insert sequenced. The full-length arginase cDNA was subsequently assembled in pArg440 and moved into pET23a for heterologous expression. An interesting feature of this strategy was not to stoichiometrically incorporate the oligonucleotide megaprimer, but use it only as an early template. This OE-PCR strategy to utilize long single-stranded megaprimer may prove handy in terms of efficiency, yield and sequence choice.

The proapoptotic activity of C-terminal domain of apoptosis-inducing factor (AIF) is separated from its N-terminal

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that mediates both NADH-oxidizing and caspase-independent apoptosis. Further, the proapoptotic activity of AIF is located in the C-terminus of AIF, although the precise minimum sequence responsible for apoptosis induction remains to be investigated. In the present study, we generated two truncated AIFs, AIFΔ1-480-FLAG, which is a FLAG-tagged C-terminal peptide comprising amino acids from 481 to 613, and AIF360-480 containing amino acids from 360 to 480 of AIF. We used confocal microscopy to demonstrate that both the truncated proteins are expressed and located in the cytoplasm of transfected cells. AIFΔ1-480 but not AIF360-480 induces apoptosis in transfected cells. We also found that the expression of AIFΔ1-480 could initiate the release of cytochrome c from the mitochondria. The suppression of caspase-9 via siRNA blocked the proapoptotic activity of AIFΔ1-480. Therefore, AIFΔ 1-480 is sufficient for inducing caspase-9-dependent apoptotic signaling, probably by promoting the release of cytochrome c. At last, we generated a chimeric immuno-AIFΔ 1-480 protein, which comprised an HER2 antibody, a Pseudomonas exotoxin A translocation domain and AIFΔ 1-480. Human Jurkat cells transfected with the immuno-AIFΔl-480 gene could express and secrete the chimeric protein, which selectively recognize and kill HER2-overexpressing tumor cells. Our study demonstrates the feasibility of the immuno-AIFΔl-480 gene as a novel approach to treating HER2-overexpressing cancers.

Cloning and characterization of drought responsive partial gene sequence(s) from Oryza sativa L. subsp. Indica.

Differential display gels were run for the drought tolerant (N-22) and drought susceptible (Panidhan) genotypes of rice (Oryza sativa) to identify the genes showing differential expression with respect to moisture stress. Differential cDNA products were cloned in PCR-Trap vector and analyzed for differential expression by Northern hybridization. Two clones namely R4A and R7G were found to be associated with water deficit stress (WDS). Sequencing revealed an insert of 244 bp in the clone R4A. BLASTN and FASTA results showed that R4A had maximum homology with a full-length cDNA clone: 002-110-H10 and OSJNBa006109.12 protein. GO classification suggested that it had beta-glucosidase motif which had been implicated in ABA mobilization and thereby ABA dependent gene expression.

Expression of Twist gene in human hepatocellular carcinoma cell strains of different metastatic potential / 华中科技大学学报（医学）（英德文版）

In order to investigate the role of Twist gene in the metastasis of hepatocellular carcinoma (HCC), total RNA was respectively extracted from three HCC cell strains with different metastatic potentials, HepG2, MHCC-97L and MHCC-97H. The first strand cDNA was synthesized by reverse transcription, which was then used as template to perform fluorescent quantitative polymerase chain reaction (FQ-PCR). The quantity of Twist gene expression was normalized by that of the housekeeping gene, GAPDH for each sample. ANOVA was used to estimate the relationship between Twist gene and metastasis potential of HCC. The results showed that the normalized initial cDNA concentrations of Twist gene in HepG2, MHCC-97L and MHCC-97H were (9.45+/-0.25)x10(-4), (1.82+/-0.41)x10(-3), (3.06+/-0.62)x10(-3), respectively. FQ-PCR revealed significant differences in the expression level of Twist among HCC cell strains with different metastatic potentials. It was concluded that high expression level of Twist was closely associated with more aggressive behaviors of HCC. Twist provides a novel indicator for HCC metastasis.

Isolation of stress responsive Psb A gene from rice (Oryza sativa l.) using differential display.

Differential display (DD) experiments were performed on drought-tolerant rice (Oryza sativa L.) genotype N22 to identify both upregulated and downregulated partial cDNAs with respect to moisture stress. DNA polymorphism was detected between drought-stressed and control leaf tissues on the DD gels. A partial cDNA showing differential expression, with respect to moisture stress was isolated from the gel. Northern blotting analysis was performed using this cDNA as a probe and it was observed that mRNA corresponding to this transcript was accumulated to high level in rice leaves under water deficit stress. At the DNA sequence level, the partial cDNA showed homology with psb A gene encoding for Dl protein.

Optimization of RQRT-pCR protocols to measure beta-1,3-glucanase mRNA levels in infected tissues of rubber tree (Hevea brasiliensis).

RQRT-PCR technique was evaluated for its validity as an alternative to Northern blotting for quantification of plant gene expression in diseased tissues of Hevea. Reliable RT-PCR results could be obtained by co-amplification of housekeeping actin gene as the internal control along with the gene of interest. The product of interest was quantified relative to that of the internal control by measuring net intensity of bands. Expression levels of defense-related beta-1,3-glucanase gene was studied in the pathogen infected tissues of rubber. The beta-1,3-glucanase gene was found to be induced in infected leaf tissues and reached a peak at 48 h after inoculation. The beta-1,3-glucanase gene expression during pathogen infection was determined through Northern blot hybridization also, using 18S RNA as the internal control. RQRT-PCR and Northern hybridization showed almost similar results, thereby validating the use of this technique to study the gene expression in rubber.

Restriction analysis of conserved and variable regions of VP2 gene of Indian isolates of bluetongue virus serotype 1.

Bluetongue virus (BTV) is a member of Orbivirus genus in family Reoviridae. The virus genome is composed of 10 double-stranded RNA segments. The RNA segment L2 encodes an outer capsid viral protein VP2, which is the main determinant of neutralization and serotype-specific immune response. BTV serotype 1 (BTV-1) specific novel primer pair was designed using VP2 gene sequences available in GenBank to amplify 1240-1844 bp region because two hypervariable and three conserved regions have been reported within these 604 nucleotides. This primer pair successfully amplified cell culture adapted six Indian isolates of BTV-1 from different geographical regions of the country. The 604 bp PCR product of VP2 gene of all six BTV-1 yielded two fragments of 273 and 331 bp when digested with Taq1 restriction enzyme. This indicated that there is only one TaqI site at 1513 bp (within 1240-1844 bp region) of VP2 gene of BTV-1 Indian isolates. The in silico restriction analysis revealed that in BTV-1 South African isolate (BTV-1SA) there is no TaqI site while in BTV-1 Australian isolates (BTV-1AUS), there are two TaqI sites (at 1513 and 1567 bp) within 1240-1844 bp region of VP2 gene. The earlier reported VP2 gene based primer pair for BTV-1 was used in the present study to amplify 2242-2933 bp region of six BTV-1 Indian isolates as three conserved regions have been reported within these 691 nucleotides. The digestion of 691 bp PCR products with XmnI yielded three fragments of 364, 173 and 154 bp with all the six Indian isolates of BTV-1 suggesting that there are two XmnI sites within 2242-2933 bp region of VP2 gene. A single XmnI site was observed in silico in BTV-1AUS and BTV-1SA isolates at different positions within this region. The in vitro and in silico restriction profile analyses of partial VP2 gene sequences using TaqI and XmnI restriction enzymes indicated a close relationship of Indian isolates of BTV-1 with BTV-1AUS isolates but not with BTV-1SA isolate.

Gene Expression Analysis of Cultured Amniotic Fluid Cell with Down Syndrome by DNA Microarray

Complete or partial triplication of human chromosome 21 results in Down syndrome (DS). To analyze differential gene expressions in amniotic fluid (AF) cells of DS, we used a DNA microarray system to analyze 102 genes, which included 24 genes on chromosome 21, 28 genes related to the function of brain and muscle, 36 genes related to apoptosis, 4 genes related to extracellular matrix, 8 genes related to other molecular function and 2 house-keeping genes. AF cells were collected from 12 pregnancies at 16-18 weeks of gestation in DS (n=6) and normal (n=6) subjects. Our DNA microarray experiments showed that the expressions of 11 genes were altered by at least 2-folds in DS, as follows. Ten genes, COL6A1, CASP5, AKT2, JUN, PYGM, BNIP1, OSF-2, PRSS7, COL3A1, and MBLL were down-regulated and GSTT1 was only up-regulated. The differential expressions of GSTT1 and COL3A1 were further confirmed by semi-quantitative RT-PCR for each sample. The gene dosage hypothesis on chromosome 21 may explain the neurological and other symptoms of DS. However, our results showed that only two genes (COL6A1 and PRSS7), among 24 genes on chromosome 21, were down-regulated in the AF cells of DS. Our data may provide the basis for a more systematic identification of biological markers of fetal DS, thus leading to an improved understanding of pathogenesis for fetal DS.

Isolation and characterization of a new mannose-binding lectin gene from Taxus media.

In this paper, we report the cloning and characterization of the first mannose-binding lectin gene from a gymnosperm plant species, Taxus media. The full-length cDNA of T. media agglutinin (TMA) consisted of 676 bp and contained a 432 bp open reading frame (ORF) encoding a 144 amino acid protein. Comparative analysis showed that TMA had high homology with many previously reported plant mannose-binding lectins and that tma encoded a precursor lectin with a 26-aa signal peptide. Molecular modelling revealed that TMA was a new mannosebinding lectin with three typical mannose-binding boxes like lectins from species of angiosperms. Tissue expression pattern analyses revealed that tma is expressed in a tissue-specific manner in leaves and stems, but not in fruits and roots. Phylogenetic tree analyses showed that TMA belonged to the structurally and evolutionarily closely related monocot mannose-binding lectin superfamily. This study provides useful information to understand the molecular evolution of plant lectins.

Molecular characterization of echovirus 11 isolates from India.

BACKGROUND & OBJECTIVES: Echovirus 11 (ECV11) is one of the most frequent non-polio enteroviruses isolated from stool samples of children with acute flaccid paralysis in north India. The present work was undertaken to study the sequence variability in the 440 bp of 5'-non-translated region of ECV11 genome using heteroduplex mobility assay (HMA). METHODS: Twelve ECV11 isolates were studied for sequence variability in the 5'-non-translated region (5'NTR) using the HMA followed by nucleotide sequencing. HMA was used to determine sequence diversity between Indian ECV11 isolates and prototype Gregory strain. HMA results were confirmed by 5'NTR nucleotide sequencing of five Indian ECV11 isolates. RESULTS: HMA results showed high genomic diversity between the prototype Gregory strain and Indian ECV11 isolates. All isolates were grouped into five different types of heteroduplex mobility patterns with respect to Gregory strain. A 440 bp 5'NTR fragment of five ECV11 isolates representing different heteroduplex patterns, was sequenced. The sequence alignment showed that 5'NTR of Indian isolates was different from prototype Gregory strain and identical to the ECV11 isolates of Finland and Hungary. Phylogenetic analysis including ECV11 isolate sequences from different parts of the world showed that Indian ECV11 isolates represented a different subgroup. INTERPRETATION & CONCLUSION: The results of the present study suggested that the HMA could be successfully used as a preliminary screening method for sequence variability determination of enterovirus field isolates. The sequence data generated on ECV11 isolates from India will be useful for future studies of endemic genotypes of echovirus.

A novel method using edge detection for signal extraction from cDNA microarray image analysis

Gene expression analyses by probes of hybridization from mRNA to cDNA targets arrayed on membranes or activated glass surfaces have revolutionized the way of profiling mega level gene expression. The main remaining problems however are sensitivity of detection, reproducibility and data processing. During processing of microarray images, especially irregularities of spot position and shape could generate significant errors: small regions of signal spots can be mis-included into background area and vice versa. Here we report a novel method to eliminate such obstacles by sensing their edges. Application of edge detection technology on separating spots from the background decreases the probability of the errors and gives more accurate information about the states of spots such as the pixel number, degree of fragmentation, width and height of spot, and circumference of spot. Such information can be used for the quality control of cDNA microarray experiments and filtering of low quality spots. We analyzed the cDNA microarray image that contains 10,368 genes using edge detection and compared the result with that of conventional method which draws circle around the spot.

Enhanced expression of a calcium-dependent protein kinase from the moss Funaria hygrometrica under nutritional starvation.

Among the downstream targets of calcium in plants, calcium-dependent protein kinases (CDPKs) form an interesting class of kinases which are activated by calcium binding. They have been implicated in a diverse array of responses to hormonal and environmental stimuli. In order to dissect the role of CDPKs in the moss Funaria hygrometrica, a polymerase chain reaction (PCR)-based approach was adopted to clone the gene. Using degenerate PCR primers against conserved regions of CDPKs, a 900 bp amplicon was obtained from the genomic DNA of Funaria. Southern hybridization under low stringency conditions indicated the presence of several CDPK related sequences in the Funaria genome. This observation is consistent with reports of multigene families of CDPKs in other plants. The 900 bp fragment was subsequently used to isolate a 2.2 kb partial genomic clone of the CDPK gene from Funaria. The genomic clone encodes an open reading frame (ORF) of 518 amino acids. Interestingly, unlike other CDPK genes from plants, the entire 1.5 kb ORF is not interrupted by introns. The deduced amino acid sequence of the Funaria gene shows extensive homology with CDPKs from higher plants, 73% identity with the Fragaria CDPK and 71% identity with CDPK isoform 7 of Arabidopsis. Phylogenetic analysis revealed that the Funaria CDPK is closer to the CDPKs from higher plants like strawberry and Arabidopsis as compared to those from lower plants such as the liverwort Marchantia, the green alga Chlamydomonas or another moss Tortula. Northern analysis shows enhanced expression of the CDPK transcript within 24-48 h of starvation for nitrogen, phosphorus or sulphur. So far the only other kinase which is known to be induced by nutrient starvation in plants is the wpk 4 which is a snf-1 related kinase (SnRKs). To our knowledge this is the first report that implicates a CDPK in the starvation response.