Abdelaleim Ismail ElSayed

Lectin approaches for glycoproteomics in FDA-approved cancer biomarkers

The nine FDA-approved protein biomarkers for the diagnosis and management of cancer are approaching maturity, but their different glycosylation compositions relevant to early diagnosis still remain practically unexplored at the sub-glycoproteome scale. Lectins generally exhibit strong binding to specific sub-glycoproteome components and this property has been quite poorly addressed as the basis for the early diagnosis methods. Here, we discuss some glycoproteome issues that make tackling the glycoproteome particularly challenging in the cancer biomarkers field and include a brief view for next generation technologies.

Preferential Lectin Binding of Cancer Cells upon Sialic Acid Treatment Under Nutrient Deprivation

The terminal monosaccharide of glycoconjugates on a eukaryotic cell surface is typically a sialic acid (Neu5Ac). Increased sialylation usually indicates progression and poor prognosis of most carcinomas. Here, we utilize two human mammary epithelial cell lines, HB4A (breast normal cells) and T47D (breast cancer cells), as a model system to demonstrate differential surface glycans when treated with sialic acid under nutrient deprivation. Under a starved condition, sialic acid treatment of both cells resulted in increased activities of α2→3/6 sialyltransferases as demonstrated by solid phase assay using lectin binding. However, a very strong Maackia amurensis agglutinin I (MAL-I) staining on the membrane of sialic acid-treated T47D cells was observed, indicating an increase of Neu5Acα2→3Gal on the cell surface. To our knowledge, this is a first report showing the utility of lectins, particularly MAL-I, as a means to discriminate between normal and cancer cells after sialic acid treatment under nutrient deprivation. This method is sensitive and allows selective detection of glycan sialylation on a cancer cell surface.

Investigation of ORF0 as a sensitive alternative diagnostic segment to detect Sugarcane yellow leaf virus

The worldwide distribution of Sugarcane yellow leaf virus (SCYLV) has led several research groups to study the function of the viral genome, the role of open reading frames (ORFs), their influence on virus accumulation and methods for diagnosis. The detection of SCYLV is usually based on the viral coat protein whether using serological and/or molecular techniques. In this study, ORF0 has been used as a diagnostic segment for SCYLV due to its highly conserved region in all SCYLV isolates. The results revealed that, ORF0 was expressed more consistently in all cultivars. In contrast, the expression of the coat protein varied. The RNA poleroviruses sequences of ORF0 were variable compared with the same segment of SCYLV populations. Analysis of the amino acid sequence of the ORF0 translation product revealed the presence of a potential transmembrane domain. The relatively high content of hydrophobic amino acids in the ORF0 protein further suggests that it may serve as a membrane anchor for the replication complex.

Biology and management of sugarcane yellow leaf virus: an historical overview

Sugarcane yellow leaf virus (SCYLV) is one of the most widespread viruses causing disease in sugarcane worldwide. The virus has been responsible for drastic economic losses in most sugarcane-growing regions and remains a major concern for sugarcane breeders. Infection with SCYLV results in intense yellowing of the midrib, which extends to the leaf blade, followed by tissue necrosis from the leaf tip towards the leaf base. Such symptomatic leaves are usually characterized by increased respiration, reduced photosynthesis, a change in the ratio of hexose to sucrose, and an increase in starch content. SCYLV infection affects carbon assimilation and metabolism in sugarcane, resulting in stunted plants in severe cases. SCYLV is mainly propagated by planting cuttings from infected stalks. Phylogenetic analysis has confirmed the worldwide distribution of at least eight SCYLV genotypes (BRA, CHN1, CHN3, CUB, HAW, IND, PER, and REU). Evidence of recombination has been found in the SCYLV genome, which contains potential recombination signals in ORF1/2 and ORF5. This shows that recombination plays an important role in the evolution of SCYLV.

Maize (Zea mays L.) constitutes a novel host to Sugarcane yellow leaf virus

Abstract Sugarcane yellow leaf virus (SCYLV) is a Polerovirus of the Luteoviridae family. In this study, SCYLV was transmitted by sugarcane aphids, Melanaphis sacchari, to two maize (Zea mays L.) lines. Infection of the maize plants was confirmed using RT-PCR with diagnostic primers YLS111 and YLS462, which produced the expected 351 bp band. The RT-PCR results were further confirmed by northern blot analysis, which revealed an accumulation of SCYLV at high levels in both maize lines. The northern blot analysis using RNA isolated from virus-infected maize plants also revealed that the genome of SCYLV is divided and contains genomic RNA (gRNA) and two subgenomic RNAs (sgRNAs) as observed in sugarcane, with estimated sizes of 6.0, 2.4 and 1.0 kb, respectively. The virus titre increased rapidly in the internodes of sugarcane cultivar H87-4094 from internode #1 (immature) to #7 (mature). This demonstrates the first successful transmission of Sugarcane yellow leaf virus by M. sacchari to maize plants.

Metabolite profiling at the cellular and subcellular level reveals metabolites associated with salinity tolerance in sugar beet

The use of non-aqueous fractionation of leaf samples with organic solvents allows differences in subcellular localization of metabolites to be determined in response to salinity stress in sugar beet.

Carbohydrate composition of sugarcane cultivars that are resistant or susceptible to Sugarcane yellow leaf virus

Sugarcane cultivars with a high (susceptible cultivars) and low (resistant cultivars) virus titer of Sugarcane yellow leaf virus were grown in the field. The carbohydrate composition in green leaf tops and in stems was determined. In RT-PCR of leaf extracts, susceptible cultivars had a high SCYLV-titer, whereas resistant cultivars had a very low titer. The cultivars differed in biomass yield, but these differences were not correlated with susceptibility. However, carbohydrate composition did have susceptibility-specific differences. Hexose levels were lower in green leaf tops and stalks of susceptible (strongly infected) cultivars than in those of resistant (weakly infected) cultivars. The stalks of susceptible cultivars also had less starch than those of resistant cultivars. Thus, the viral susceptibility (and infection) affected sugar metabolism. In addition, a positive correlation between hexose and starch in stems and between hexose and sucrose in green leaf tops was observed. The results from susceptible versus resistant cultivars were the opposite of those in the comparison between infected versus virus-free lines of the same cultivar. The breeding process apparently had unintentionally selected clones with modulated carbohydrate metabolism to avoid or compensate for the adverse effects of SCYLV infection.

Molecular marker assisted for recognition drought tolerant in some of bread wheat genotypes

To develop crop plants with enhanced tolerance of drought stress, a basic understanding of physiological, biochemical, and genetic networks is essential. Four bread wheat genotypes and one wheat line were evaluated for molecular indicators of drought tolerance using RAPD-PCR and protein profiling. The RAPD markers were used to determine the genetic differences between the five wheat genotypes and to determine the molecular markers associated with tolerance to drought. The present study found that RAPD analysis is a valuable diagnostic tool when different sets of RAPD primers were used to study the polymorphism at the molecular level. A total of 72 alleles were amplified with six random primers out of which 61% were monomorphic and 38% were polymorphic. Primer B8 amplified a 600 bp band in Sham-6 which is assumed to be a drought-tolerant genotype, while primer A-8 amplified a 550 bp band in genotypes Giza-168 and Sham-6. Genetically, the most similar genotypes were Sham-6 and Line-7 (93%) followed by Gemaza-9 and Giza-168 (92%) while the most dissimilar genotype was Sakha-93 (86%). Protein profiling revealed differences between the genotypes with a protein band presents at 130 KDa in the Sham-6, Gemaza-9, and Sakha-93 genotypes and absent in Line-7 and Giza-168. Proline content was highest in the drought-tolerant genotypes, Sham-6 and Sakha-93. Sucrose content in shoots was increased in tolerant plants (Sakha-93 and Sham-6), while there was a reduction in sucrose in the shoot tissues of the seedling stage of Gemaza-9, Line-7, and Giza-168. Overall, the accumulation of reducing sugars was lowest in all plants compared with sucrose content.

Molecular and biochemical characterisation of a novel type II peroxiredoxin (XvPrx2) from the resurrection plant Xerophyta viscosa

A type II peroxiredoxin gene (XvPrx2) was isolated from a Xerophyta viscosa (Baker) cDNA cold-stress library. The polypeptide displayed significant similarity to other plant type II peroxiredoxins, with the conserved amino acid motif (PGAFTPTCS) proposed to constitute the active site of the enzyme. Northern blot analyses showed that XvPrx2 gene was stress-inducible in response to abiotic stresses while gel analyses revealed that XvPrx2 homologues exist within the X. viscosa proteome. Using a yellow fluorescent reporter protein, the XvPrx2 protein localised to the cytosol. A mutated protein (XvV7) was generated by converting the valine at position 76 to a cysteine and an in vitro DNA protection assay showed that, in the presence of either XvPrx2 or XvV7, DNA protection occurred. In addition, an in vivo assay showed that increased protection was conferred to Escherichia coli cells overexpressing either XvPrx2 or XvV7. The XvPrx2 activity was maximal with DTT as electron donor and H2O2 as substrate. Using E. coli thioredoxin, a 2-15-fold lower enzyme activity was observed. The XvPrx2 activity with glutathione was significantly lower and glutaredoxin had no measurable effect on this reaction. The XvV7 protein displayed significantly lower activity compared with XvPrx2 for all substrates assessed.

Enhancing antioxidant systems by exogenous spermine and spermidine in wheat (Triticum aestivum) seedlings exposed to salt stress

Plants have evolved complex mechanisms to mitigate osmotic and ionic stress caused by high salinity. The effect of exogenous spermine (Spm) and spermidine (Spd) on defence responses of wheat seedlings under NaCl stress was investigated by measuring antioxidant enzyme activities and the transcript expression of corresponding genes. Exogenous Spm and Spd decreased the level of malondialdehyde, increased chlorophyll and proline contents, and modulated PSII activity in wheat seedlings under salt stress. Spermidine alleviated negative effects on CO2 assimilation induced by salt stress in addition to significantly increasing the activity and content of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). It appears Spd conferred salinity tolerance in wheat seedlings by enhancing photosynthetic capacity through regulation of gene expression and the activity of key CO2 assimilation enzymes. Exogenous Spm regulated activities of different antioxidant enzymes (catalase, glutathione reductase, dehydroascorbate reductase, ascorbate peroxidase, and superoxide dismutase) and efficiently modulate their transcription levels in wheat seedlings under salt stress. It is likely that Spm plays a key role in alleviating oxidative damage of salt stress by adjusting antioxidant enzyme activities in plants. In addition, exogenous Spd increased transcript level of spermine synthase under salt stress. Salinity stress also caused an increase in transcript levels of diamine oxidase (DAO) and polyamine oxidase (PAO). Exogenous Spd application resulted in a marked increase in free Spd and Spm contents under saline conditions. These results show that exogenous Spd and Spm effectively upregulated transcriptional levels of antioxidant enzyme genes and improved the defence response of plants under salt stress.