Wild Clones Research Articles

Colorimetric Analysis, Genetic Control, and Effects on Wood Properties of Green Vein in Wild Cherry

Green vein is one of the most severe defects that affect wild cherry (Prunus avium L.). It consists of green streaks that alter the typical color and uniformity of the cherry wood, causing considerable value losses. A colorimetric analysis was performed on wild cherry clones using the CIE L*a*b* system, and the influences of environmental and genetic factors on green vein as well as the effects of the presence of green vein on the physical properties of the wood were investigated. Discriminant analysis shows that the color parameter that best discriminated green vein were low values of L* and a*. The cloning effect was the most important, but the environment also played an important role in the development of green vein. Finally, the presence of green vein was found to mainly affect the longitudinal shrinkage of wood and, to a lesser extent, wood density. These same features are typical of tension wood, to which green vein was strictly linked, as confirmed by some preliminary anatomical observations.

Small molecules mediate bacterial farming by social amoebae

The origin of domestication persists as an important question at the nexus between biology, archaeology, and history (1). As intensive research continues apace on organisms whose domestication accompanied the emergence of modern human societies (2, 3), the study of agricultural evolution has expanded to include nonhuman organisms (bark beetles, termites, ants) whose natural history is interwoven with the dispersal, cultivation, and sustainable harvest of microbial symbionts (4). The most recent addition to the roster of nonhuman farmers is Dictyostelium discoideum , a social amoeba that preys upon soil bacteria (5, 6). The life cycle of D. discoideum makes it a favorable model system for the study of cooperation, altruism, and other aspects of social evolution (7⇓–9). When food is scarce, single-celled amoebae aggregate into a multicellular “slug,” which eventually forms a stalked fruiting body from which spores disperse to new habitats. Up to a third of all wild clones of D. discoideum are “farmer strains” that incorporate bacteria into fruiting bodies and establish new food crops during spore dispersal (5), using, as it were, a “take out” foraging strategy that confers advantages under conditions of food scarcity. However, only half of the bacterial cells ( Pseudomonas fluorescens ) carried by farmer strains of D. discoideum are eaten, begging a closer investigation of the bacterial crop to determine whether its domestication reflects hidden costs and benefits of its association with social amoebae. In PNAS, Stallforth et … [↵][1]1E-mail: rar229{at}cornell.edu. [1]: #xref-corresp-1-1

Abstract 155: Mutations In ABCA8 Result In HDL Deficiency And Cholesterol Efflux Defects

The molecular basis of extreme HDL-C levels is incompletely understood. To identify mutations in novel putative HDL genes, we sequenced select genes in 80 unrelated individuals with extreme low HDLc (HDL %ile≤10th) and as controls, 120 individuals with high HDL (HDL %ile≥90th). We identified two variants in ABCA8 in subjects with low HDL-C levels. The E18-2T>C mutation is located in a highly conserved region and gives rise to a disruption of a splice site. The G>C variant occurs at a site conserved in vertebrates, at position 64425884 (build HG18), and results in Pro609Arg substitution. No ABCA8 mutations were identified in HHDL individuals. These represent the first mutations identified in ABCA8, which belongs to the family of ATP binding cassette transporters. Family expansion followed by genotyping identified additional mutation carriers and first degree relative controls in whom segregation analyses were performed. Compared with controls, mutation carriers showed a significant 32.6% decrease in plasma HDLc levels and decreased HDL percentiles (HDLc: controls = 1.23±0.26, mutation carriers = 0.83±0.37, mmol/L, p=0.0007; HDL%ile: controls = 41.8±21.2, mutation carriers = 17.3±19.2, p=0.004, n=23 controls and 12 carriers). No changes in LDLc, triglycerides or BMI were observed. Since mutations in ABCA8 reduce plasma HDLc levels, we assessed the role of ABCA8 in lipid efflux. Cholesterol efflux to lipid-free APOA-I was significantly reduced by 62.1% in fibroblasts from the proband with the E18-2 T>C mutation. In comparison, fibroblasts from heterozygous ABCA1 mutation carriers showed a 30-60% decrease in efflux to APOA-I depending on mutation severity. Wild type and P609R ABCA8 cDNA clones were generated from human liver RNA. A significant ~200% increase in cholesterol efflux to lipid free APOA-I was observed with wild type ABCA8, further suggesting that ABCA8 is a lipid transporter. A significant 75% decrease in the efflux of cholesterol to lipid-free APOA-I (p=0.02) was observed with the mutant P609R ABCA8 protein compared to wild-type. We show here that ABCA8 is a cholesterol transporter, and that mutations in ABCA8 are a novel cause for reduced plasma HDLc levels in humans.

RESPONSES IN NITROGEN MASS AND NITROGEN METABOLISM OF WILD SUGARCANE (<I>SACCHARUM SPONTANEUM</I> L.) CLONES TO ENHANCED UV-B RADIATION UNDER FIELD CONDITIONS

Field experiments were conducted to evaluate effects of enhanced UV-B radiation (280-315 nm) on intraspecific responses in nitrogen mass and nitrogen metabolism of six wild sugarcane (Saccharum spontaneum L.) clones. The clones were collected from original sites with different altitudes (4-1780 m above sea-level) and latitudes (18-38°N). The supplemental UV-B radiation was 5.00 kJ⋅m-2, simulating a depletion of 20% stratospheric ozone. Out of the six tested wild sugarcane clones, available Nitrogen (N) contents in soil and N contents in leaves of four wild sugarcane clones decreased significantly. Leaf biomass and leaf N mass of six wild sugarcane clones significantly increased. UV-B radiation significantly decreased N contents and increased biomass and N mass in stems of six wild sugarcane clones. Contents of free amino acid of two wild sugarcane clones (92-11 and 92-36) significantly decreased, whilst that of clones 83-193 and II91-5 significantly increased. Total protein contents of three wild sugarcane clones significantly decreased, whilst that of clone 90-15 significantly increased. Nitrate reductase activities of two clones (92-11 and 90-15) significantly decreased, whilst that of four clones significantly increased. Glutamine synthetase activities of two clones (92-11 and 90-15) significantly increased, while that of clones 83-193 significantly decreased. UV-B radiation might change stem biomass, resulting in changes in N mass in stem and leaves. Results indicate that intraspecific responses in N mass and N metabolism of six wild sugarcane (S. spontaneum) clones differing in UV sensitivity to enhanced UV-B radiation exist under field conditions.

A SBP-Box Gene VpSBP5 from Chinese Wild Vitis Species Responds to Erysiphe necator and Defense Signaling Molecules

A novel SQUAMOSA PROMOTER BINDING PROTEIN (SBP)-box gene, designated as VpSBP5, was isolated from Chinese wild Vitis pseudoreticulata clone “Baihe-35-1” infected by Erysiphe necator under natural field conditions. The full-length cDNA sequence of VpSBP5 comprised 3,811 bp and encoded a polypeptide of 1,030 amino acids which contained a highly conserved SBP domain bearing two zinc-binding sites of the C2HCH type and a nuclear localization signal. The deduced amino acid sequence of VpSBP5 was identical to that of VvSBP5 and grouped into the same clade (group 1) with AtSPL1, AtSPL12, AtSPL14, VvSBP7, and VvSBP17. We further confirmed that VpSBP5 gene is indeed targeted to the nucleus and possesses transcriptional activation activity. The expression of VpSBP5 as determined by reverse transcription PCR, was induced by E. necator in the E. necator-resistant V. pseudoreticulata clone Baihe-35-1 and the susceptible clone “Hunan-1,” and exhibited a quicker response in Baihe-35-1. Moreover, the expression of VpSBP5 was induced by salicylic acid (SA) and methyl jasmonate (MeJA) in Baihe-35-1. Together, our results indicate that VpSBP5 is likely to participate in the regulation of the resistance to E. necator by inducing SA and MeJA molecular signals in grape, and the degree of disease resistance of the grapevine genotypes may correlate with the time of the peak appearing.

Host Races of the Cotton Aphid,Aphis gossypii, in Asexual Populations from Wild Plants of Taro and Brinjal

Worldwide, several studies have shown that adaptation to different host plants in phytophagous insects can promote speciation. The cotton aphid, Aphis gossypii Glover (Homoptera: Aphididae: Aphidini), is a highly polyphagous species, but its populations increase by parthenogenetic reproduction alone in Indian subcontinent. This study showed that genotypes living in wild plants of taro, Colocasia esculenta var. esculenta (L.) Schott (Alismatales: Araceae), and brinjal, Solanum torvum Swartz (Solanales: Solanaceae), behave as distinct host races. Success rates of colonization after reciprocal host transfers were very poor. Clones of A. gossypii from wild taro partly survived in the first generation when transferred to wild brinjal, but nymph mortality was 100% in the second generation. In contrast, brinjal clones, when transferred to taro, could not survive even in the first generation. Significant differences between the clones from two host species were also recorded in development time, generation time, fecundity, intrinsic rate of increase, net reproductive rate, and mean relative growth rate. Morphologically, aphids of wild taro clones possessed longer proboscis and fore-femora than the aphids of the brinjal clones. The results showed that A. gossypii exists as distinct host races with different abilities of colonizing host plants, and its populations appear to have more potential of sympatic evolution than previously regarded.

Expression of a GDP-L-galactose phosphorylase-like gene in a Chinese wild Vitis species induces responses to Erysiphe necator and defense signaling molecules

Using rapid amplification of cDNA ends, a full-length cDNA sequence of a GDP-L-galactose phosphorylase-like gene was isolated from leaves infected by Erysiphe necator in the Chinese wild (Vitis pseudoreticulata) clone, 'Baihe-35-1', an E. necator-resistant genotype. The full-length cDNA, designated as VpVTC, comprised 1943 bp and putatively encodes a 453-amino acid polypeptide containing an HIT motif. The deduced amino acid sequence showed high similarity with that of VTC genes from other plants. The expression of VpVTC, determined by reverse transcriptase-polymerase chain reaction, was induced by E. necator and defense signaling molecules, including salicylic acid, methyl jasmonate, and ethephon, in 'Baihe-35-1', the V. quinquangularis genotype 'Shang-24', and the E. necator-susceptible V. pseudoreticulata genotype, 'Hunan-1'. Transcript levels of VpVTC correlated well with the degree of disease resistance in the 3 genotypes. Maximum induction of VpVTC by E. necator (>7-fold at 96 h post-inoculation) occurred in 'Baihe-35-1', which also showed the fastest response to signaling molecules. Upregulating the expression of VpVTC in 'Baihe-35-1' resulted in a gradual increase in the ascorbic acid concentration of leaves inoculated with E. necator. Furthermore, VpVTC was expressed in leaves, stems, inflorescence, tendrils, and fruit at all developmental stages, with the highest level occurring in fruit 35 days after flowering.

Clonal variation in Scots pine (Pinus sylvestris) and in transgenic silver birch (Betula pendula)

The aim of this study is to assess the clonal variation in Scots pine (Pinus sylvestris L.) clones and in transgenic lines of silver birch (Betula pendula Roth) and its causes, with special attention to the effects of cloning and transgenesis in tree breeding programmes. The parental effect on cloning success variation was studied in an experiment where Scots pine embryogenic lines were initiated from immature seeds of a full diallele cross. The evaluation was made after culture initiation, on maintenance medium and by mature embryo production. Growth and stem straightness of Scots pine clones were assessed in a 10-year field trial established with rooted cuttings. The effect of a single gene transfer, the sugar beet chitinase IV gene (chiIV), was assessed on plant growth, susceptibility to fungal diseases and development of root associated fungal communities and phenology, in a 3-year field trial established with micropropagated transgenic silver birch lines and wild-type clones. The results of the somatic embryogenesis experiment with Scots pine showed that the initiation success, as well as maturation, was more affected by the genotype of the mother than the one of the father, while during the proliferation period the mother’s effect decreased and the father’s increased. The field trial with Scots pine showed that the tree’s genotype, more than the propagation method, has an effect on the plant behaviour in the field. In silver birch transgenic lines, the introduction of a single gene (chiIV) led to a reduction in growth and quality characteristics, although no significant changes occurred regarding fungal disease resistance, ectomycorrhizal colonization or fungal community structure, as compared to the natural variation occurring in wild type clones. The conclusion was that the variation in the success of Scots pine embryogenesis and in the growth of Scots pine rooted cuttings is strongly affected by genotype and, hence, the rooted cuttings are suitable for testing height growth in breeding programmes. A single chiIV gene transfer did not improve significantly fungal disease resistance in silver birch or interfere with root associated fungal community. However, the variation in adaptive traits among silver birch transgenic lines is at the same level with the variation detected in randomly selected wild-type silver birches and single transgenic lines may be selected in breeding programmes.

Abstract P4-06-03: Zinc Finger Nuclease Genome Engineering Reveal Multiple Functions of Secretory Leukocyte Peptidase Inhibitor in Regulating Pleuripotency of Cancer Stem Cells in Inflammatory Breast Cancer

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive and lethal variant of this disease and is known to be enriched for cells with a cancer stem cell phenotype. IBC is characterized by the presence of cell aggregates, defined as tumor emboli, that metastasize into skin and chest wall. The only documented biomarker of tumor emboli is the surface glycoprotein, E-cadherin. We previously demonstrated that IBC tumor emboli express the alarm anti-protease, secretory leukocyte peptidase inhibitor (SLPI), a metastasis related gene highly expressed in IBC patient tumors. Since the function of SLPI in IBC is unknown, the present studies used zinc finger technology to knockout (KO) copies of SLPI in the SUM149 IBC cell line to define the role of SLPI in IBC. Materials and Methods: Using CompoZr zinc finger nuclease (ZFN) technology (Sigma-Aldrich), SLPI KO cell lines were generated by disrupting all alleles (3) in exon 1 of SUM149 cells derived from an IBC primary tumor with a high CD44+cell population. The target-specific ZFNs bound DNA at a sequence-specific location and created double strand breaks repaired by non-homologous end joining, resulting in deletions at the SLPI locus. Single cell SLPI KO clones were isolated and serially passaged to establish stable cell lines. Functional assays were used to assess proliferation, invasion, tube formation and clonogenicity. Global transcriptional profiling was performed to identify genes and signaling pathways directly regulated by SLPI. Results: SUM149 SLPI KO clones did not produce SLPI protein and had a significantly slower turn-over time of 75 hrs compared with 24 hrs in SUM149 wild type clones. Loss of SLPI blocked invasion by 50%, and completely inhibited formation of tube-like structures, an activity defined as vasculogenic mimicry, characteristic of IBC. The loss of only 1 SLPI allele resulted in the inability of SUM149 cells to grow as anchorage independent clones in soft agar, commonly used as a predictor of in vivo tumorigenicity. SLPI directly regulated expression of multiple genes within the embryonic stem cell pleuripotency canonical pathway, including WNT, Frizzled, PDK-1, platelet derived growth factor receptor and sphingosine-1-phosphate receptor. Studies are underway to determine the specific role of SLPI in IBC tumor growth and metastasis. Conclusions: Our previous studies demonstrated that SLPI is expressed by IBC tumor emboli and can be used as a biomarker of tumor emboli in IBC core and skin punch biopsies. SLPI was found to not only regulate critical functional activities of IBC tumor cells but also to directly regulate genes within pathways critically important to maintenance of pleuripotency of tumors with a cancer stem cell phenotype. Collectively, these studies demonstrate the power and utility of the zinc finger technology, which enables the interrogation of tumor cells to discern the direct function and role of specific genes of interest. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-06-03.

Characterization of Erysiphe necator-responsive genes in Chinese Wild Vitis quinquangularis.

Powdery mildew (PM), caused by fungus Erysiphe necator, is one of the most devastating diseases of grapevine. To better understand grapevine-PM interaction and provide candidate resources for grapevine breeding, a suppression subtractive hybridization (SSH) cDNA library was constructed from E. necator-infected leaves of a resistant Chinese wild Vitis quinquangularis clone “Shang-24”. A total of 492 high quality expressed sequence tags (ESTs) were obtained and assembled into 266 unigenes. Gene ontology (GO) analysis indicated that 188 unigenes could be assigned with at least one GO term in the biological process category, and 176 in the molecular function category. Sequence analysis showed that a large number of these genes were homologous to those involved in defense responses. Genes involved in metabolism, photosynthesis, transport and signal transduction were also enriched in the library. Expression analysis of 13 selected genes by qRT-PCR revealed that most were induced more quickly and intensely in the resistant material “Shang-24” than in the sensitive V. pseudoreticulata clone “Hunan-1” by E. necator infection. The ESTs reported here provide new clues to understand the disease-resistance mechanism in Chinese wild grapevine species and may enable us to investigate E. necator-responsive genes involved in PM resistance in grapevine germplasm.

Amino-terminal Cysteine Residues Differentially Influence RGS4 Protein Plasma Membrane Targeting, Intracellular Trafficking, and Function

Regulator of G-protein signaling (RGS) proteins are potent inhibitors of heterotrimeric G-protein signaling. RGS4 attenuates G-protein activity in several tissues. Previous work demonstrated that cysteine palmitoylation on residues in the amino-terminal (Cys-2 and Cys-12) and core domains (Cys-95) of RGS4 is important for protein stability, plasma membrane targeting, and GTPase activating function. To date Cys-2 has been the priority target for RGS4 regulation by palmitoylation based on its putative role in stabilizing the RGS4 protein. Here, we investigate differences in the contribution of Cys-2 and Cys-12 to the intracellular localization and function of RGS4. Inhibition of RGS4 palmitoylation with 2-bromopalmitate dramatically reduced its localization to the plasma membrane. Similarly, mutation of the RGS4 amphipathic helix (L23D) prevented membrane localization and its G(q) inhibitory function. Together, these data suggest that both RGS4 palmitoylation and the amphipathic helix domain are required for optimal plasma membrane targeting and function of RGS4. Mutation of Cys-12 decreased RGS4 membrane targeting to a similar extent as 2-bromopalmitate, resulting in complete loss of its G(q) inhibitory function. Mutation of Cys-2 did not impair plasma membrane targeting but did partially impair its function as a G(q) inhibitor. Comparison of the endosomal distribution pattern of wild type and mutant RGS4 proteins with TGN38 indicated that palmitoylation of these two cysteines contributes differentially to the intracellular trafficking of RGS4. These data show for the first time that Cys-2 and Cys-12 play markedly different roles in the regulation of RGS4 membrane localization, intracellular trafficking, and G(q) inhibitory function via mechanisms that are unrelated to RGS4 protein stabilization.

Identification of genes differentially expressed in grapevine associated with resistance to Elsinoe ampelina through suppressive subtraction hybridization

Anthracnose, caused by the biotrophic fungus Elsinoe ampelina, is an economically devastating disease of grapevine (Vitis vinifera L.) prevalent in warm and humid regions of the world. In order to investigate the molecular resistance mechanisms and identify genes related to anthracnose resistance in grapevine, a Suppression Subtractive Hybridization (SSH) library was constructed using mixed cDNAs prepared from leaves of Chinese wild Vitis quinquangularis clone 'Shang-24', cDNA prepared from leaves infected with the pathogen E.ampelina served as tester and cDNA from mock-inoculated leaves as driver. A total of 670 high-quality ESTs were clustered and assembled into a collection of 461 unique genes comprising 85 contigs and 376 singletons. By Gene ontology (GO) analysis 310 unigenes were assigned to 22 GO slims within the molecular function category, while 317 unigenes could be sorted into 43 GO slims within the biological process category. The expression profiles of 20 selected genes, monitored by quantitative RT-PCR, indicated that expression of these genes in the E.ampelina-resistant 'Shang-24' was quicker and more intense, than in the susceptible 'Red Globe' where the reaction was delayed and limited. The results imply that these up-regulated genes could be involved in grapevine responses against E.ampelina infection.

Hybridization barriers between diploid Solanum tuberosum and wild Solanum raphanifolium

Wild potato germplasm represents a unique, diverse and accessible resource for disease and pest resistance, along with useful agronomic traits that may be introgressed into the cultivated potato (Solanum tuberosum L.). Hybridization of diploid wild Solanum species with haploids (2×) of cultivated potato (4×) is generally an effective technique for introducing genetic diversity and desirable traits into potato. However, in this study, hybridization barriers were found in crosses between S. tuberosum haploids and the wild species S. raphanifolium. Male sterility, likely due to nuclear-cytoplasmic interactions, was observed in some haploid tbr × S. raphanifolium hybrids. In addition, pollen-pistil incompatibilities were observed in backcross, F2, and reciprocal cross hybridization attempts that failed to produce seed. More crosses were successful when F1 clones were crossed as females to wild clones than to cultivated clones. When crosses were made in the other direction, with F1 hybrids used as male parents, seeds were almost never produced.

High relatedness in a social amoeba: the role of kin-discriminatory segregation

A major challenge for social theory is to explain the importance of kin discrimination for the evolution of altruism. One way to assess the importance of kin discrimination is to test its effects on increasing relatedness within groups. The social amoeba Dictyostelium discoideum aggregates to form a fruiting body composed of dead stalk and live spores. Previous studies of a natural population showed that where D. discoideum occurs in the soil, multiple clones are often found in the same small soil samples. However, actual fruiting bodies usually contain only one clone. We here performed experiments to gauge the effect of kin-discriminatory segregation on increasing relatedness. We mixed co-occurring clones from this population using a relatedness level found in small soil samples. We found a lower proportion of uniclonal fruiting bodies and a lower level of relatedness compared with natural fruiting bodies. We found that the amount of relatedness increase attributable to kin-discriminatory segregation was small. These findings suggest a relatively minor influence of kin-discriminatory segregation on relatedness in D. discoideum. We discuss our results comparing with the results of previous studies, including those of wild clones and laboratory mutants. We ask why wild clones of D. discoideum exhibit a low degree of kin-discriminatory segregation, and what alternative factors might account for high relatedness in D. discoideum.

The Effect of Photoperiod on Tuberisation in Cultivated × Wild Potato Species Hybrids

Wild Solanum species offer a valuable source of genetic diversity for potato improvement. Most of these species are found in equatorial South and Central America and they do not tuberise under long-day photoperiods typical of those in the major potato production areas of North America, Europe and Asia. Crosses between two haploids of Solanum tuberosum Chilotanum Group as females and two wild Solanum species clones generated four hybrid families. The parents and progeny were subjected to two greenhouse trials to evaluate tuberisation at 20-, 14-, and 8-h photoperiods. Parents and offspring tuberised most readily at the 8-h photoperiod, and poorly or not at all at the 20-h photoperiod. Segregation for tuberisation in hybrids was apparent at the 14-h photoperiod and depended on the cultivated parent, but not the wild species parent. The data support previous studies and best fit a model in which tuber production under long photoperiods is controlled by two dominant genes in cultivated × wild species hybrids.

Isolation of wild microalgae from natural water bodies for high hydrogen producing strains

Water samples from various marine, freshwater and terrestrial environments in China have been collected and screened in our laboratory for wild clones of microalgae since 2008. Hundreds of microalgal clones were purified through plate streaking, of which 52 strains have been screened for their ability to produce H2 under anaerobic conditions. Twenty eight strains including 13 freshwater green algae, 12 marine green algae and 3 cyanobacteria were found to evolve H2 under sulfur or nitrogen deprivation. Parietochloris incisa, Chlorella protothecoides, Chlorella capsulata, Nannochloropsis sp., Tetraselmis helgolandica, Pyramimonas sp., Chlorella autotrophica, Dunaliella apiculata, Tetraselmis striata and Tetraselmis tetrathele were first reported as new H2 producing species. Among those H2 producing strains, freshwater C. protothecoides showed the highest H2 producing capacity (2.93 H2 ml/l/h) and accumulated up to 123.6 ml H2 per liter culture under sulfur deprivation. Marine C. autotrophica and T. striata were two notable green marine species that produced H2 phototrophically, with 3.73 ml/l and 20.11 ml/l of H2 yield respectively. Our results also demonstrated that both the likelihood of a strain producing H2, and the average H2 output, in freshwater strains were much higher than that in marine strains. In addition, the time for inducing H2 production in freshwater strains was short compared to the marine ones. All three Arthrospira strains tested were able to generate H2 under sulfur (S-) deprivation though the cultures would quickly lose their vitality in S-deprived medium. Interestingly, Arthrospira produced H2 under nitrogen deprivation, and the H2 yield was even higher than under S-depletion. However, neither tested chrysophyte nor diatom produced any H2 under anaerobiosis caused by S-deprivation.

Identification and expression analysis of a pathogen-responsive PR-1 gene from Chinese wild Vitis quinquangularis

In response to pathogen attacks, plant produces a wide range of pathogenesis-related (PR) proteins. PR-1 genes represent the first identified PR gene family. Most members of PR-1 gene family are not inducible by pathogen attacks. In this study, we identified a pathogen-responsive PR-1 gene designated as VqPR-1 (GenBank accession no. JN256202), in a subtractive suppression hybridization (SSH) cDNAlibrary from Elsinoe ampelina -inoculated young leaves of Chinese wild Vitis quinquangularis clone ‘shang-24’. VqPR-1 protein contained the requisite signal sequence at the N-terminus, a conserved three-dimensional structure called ‘PR-1 fold’ and a highly conserved six-cysteine motif. Expression level of VqPR-1 rose rapidly in response to E. ampelina infection. The three tested plant defence signaling molecules, salicylic acid (SA), ethephon (Eth) and methyl jasmonate (MeJA) all triggered an induction of VqPR-1. However, the induction by addition of MeJA was weaker than that induced by SA and Eth. In addition, the response to inoculation with E. ampelina or treatment with signaling molecules, was sometimes a suppression of VqPR-1 gene expression. The highest expression of VqPR- 1 was observed in flowers, stems and leaves, while low-level or no obvious transcripts were detected in pericarps and tendrils, respectively. Key words: Vitis quinquangularis, PR-1 gene, Elsinoe ampelina, expression analysis.

Loss of keratin 8 phosphorylation leads to increased tumor progression and correlates with clinico-pathological parameters of OSCC patients.

BackgroundKeratins are cytoplasmic intermediate filament proteins expressed in tissue specific and differentiation dependent manner. Keratins 8 and 18 (K8 and K18) are predominantly expressed in simple epithelial tissues and perform both mechanical and regulatory functions. Aberrant expression of K8 and K18 is associated with neoplastic progression, invasion and poor prognosis in human oral squamous cell carcinomas (OSCCs). K8 and K18 undergo several post-translational modifications including phosphorylation, which are known to regulate their functions in various cellular processes. Although, K8 and K18 phosphorylation is known to regulate cell cycle, cell growth and apoptosis, its significance in cell migration and/or neoplastic progression is largely unknown. In the present study we have investigated the role of K8 phosphorylation in cell migration and/or neoplastic progression in OSCC.Methodology and Principal FindingsTo understand the role of K8 phosphorylation in neoplastic progression of OSCC, shRNA-resistant K8 phospho-mutants of Ser73 and Ser431 were overexpressed in K8-knockdown human AW13516 cells (derived from SCC of tongue; generated previously). Wound healing assays and tumor growth in NOD-SCID mice were performed to analyze the cell motility and tumorigenicity respectively in overexpressed clones. The overexpressed K8 phospho-mutants clones showed significant increase in cell migration and tumorigenicity as compared with K8 wild type clones. Furthermore, loss of K8 Ser73 and Ser431 phosphorylation was also observed in human OSCC tissues analyzed by immunohistochemistry, where their dephosphorylation significantly correlated with size, lymph node metastasis and stage of the tumor.Conclusion and SignificanceOur results provide first evidence of a potential role of K8 phosphorylation in cell migration and/or tumorigenicity in OSCC. Moreover, correlation studies of K8 dephosphorylation with clinico-pathological parameters of OSCC patients also suggest its possible use in prognostication of human OSCC.

Engineered NADH-dependent GRE2 from Saccharomyces cerevisiae by directed enzyme evolution enhances HMF reduction using additional cofactor NADPH

Furfural and 5-hydroxymethylfurfural (HMF) are inhibitors generated by lignocellulosic biomass pretreatment such as dilute acid hydrolysis that inhibit microbial growth and interfere with subsequent fermentation. It is possible to in situ detoxify these inhibitory compounds by aldehyde reductions using tolerant Saccharomyces cerevisiae. YOL151W ( GRE2) is a commonly recognized up-regulated gene expressed under stress conditions that encodes reductase activities toward furfural and HMF using cofactor NADH. Applying a directed enzyme evolution approach, we altered the genetic code of GRE2 yielding two mutants with amino acid substitutions of Gln261 to Arg261 and Phe283 to Leu283; and Ile107 to Val107, Gln261 to Arg261, and Val285 to Asp285 for strain Y62-C11 and Y62-G6, respectively. Clones of these mutants showed faster growth rates and were able to establish viable cultures under 30 mM HMF challenges when compared with a wild type GRE2 clone when inoculated into synthetic medium containing this inhibitor. Compared with the wild type control, crude cell extracts of the two mutants showed 3- to 4-fold and 3- to 9-fold increased specific enzyme activity using NADH toward HMF and furfural reduction, respectively. While retaining its aldehyde reductase activities using the cofactor NADH, mutant Y62-G6 displayed significantly greater reductase activities using NADPH as the cofactor with 13- and 15-fold increase toward furfural and HMF, respectively, as measured by its partially purified protein. Using reverse engineering and site directed mutagenesis methods, we were able to confirm that the amino acid substitution of the Asp285 is responsible for the increased aldehyde reductase activities by utilizing the additional cofactor NADPH.

Sorafenib monotherapy gives sustainable suppression of FLT3 clone in untreated patients with FLT3‐internal tandem duplication positive acute myeloid Leukaemia

Sorafenib monotherapy gives sustainable suppression of FLT3 clone in untreated patients with FLT3‐internal tandem duplication positive acute myeloid Leukaemia