Structural plasticity of peanut lectin: an X-ray analysis involving variation in pH, ligand binding and crystal structure

Until recently, it has only been possible to grow crystals of peanut lectin when complexed with sugar ligands. It is now shown that it is possible to grow peanut lectin crystals at acidic pH in the presence of oligopeptides corresponding to a loop in the lectin molecule. Crystals have also been prepared in the presence of these peptides as well as lactose. Low-pH crystal forms of the lectin-lactose complex similar to those obtained at neutral pH have also been grown. Thus, crystals of peanut lectin grown under different environmental conditions, at two pH values with and without sugar bound to the lectin, are now available. They have been used to explore the plasticity and hydration of the molecule. A detailed comparison between different structures shows that the lectin molecule is sturdy and that the effect of changes in pH, ligand binding and environment on it is small. The region involving the curved front β-sheet and the loops around the second hydrophobic core is comparatively rigid. The back β-sheet involved in quaternary association, which exhibits considerable variability, is substantially flexible, as is the sugar-binding region. The numbers of invariant water molecules in the hydration shell are small and they are mainly involved in metal coordination or in stabilizing unusual structural features. Small consistent movements occur in the combining site upon sugar binding, although the site is essentially preformed

Cancer targeting with biomolecules: a comparative study of photodynamic therapy efficacy using antibody or lectin conjugated phthalocyanine-PEG gold nanoparticles

The functionalisation of therapeutic nanoparticle constructs with cancer-specific biomolecules can enable selective tumour accumulation and targeted treatment. Water soluble gold nanoparticles (ca. 4 nm) stabilised by a mixed monolayer of a hydrophobic zinc phthalocyanine photosensitiser (C11Pc) and hydrophilic polyethylene glycol (PEG) have been prepared. The C11Pc-PEG gold nanoparticle constructs were further functionalised with jacalin, a lectin specific for the cancer-associated Thomsen–Friedenreich (T) carbohydrate antigen, or with monoclonal antibodies specific for the human epidermal growth factor receptor-2 (HER-2). The two biofunctionalised nanoparticle conjugates produced similar levels of singlet oxygen upon irradiation at 633 nm. Importantly, both nanoparticle conjugates demonstrated extensive, yet comparable, phototoxicity in HT-29 colorectal adenocarcinoma cells (80–90%) and in SK-BR-3 breast adenocarcinoma cells (>99%). Non-conjugated C11Pc-PEG gold nanoparticles were only minimally phototoxic. Lysosomal colocalisation studies performed with the HT-29 colon cancer cells and the SK-BR-3 breast cancer cells revealed that both nanoparticle conjugates were partially localised within acidic organelles, which is typical of receptor-mediated endocytosis. The similarity of the targeted PDT efficacy of the two biofunctionalised C11Pc-PEG gold nanoparticles is discussed with respect to targeting ligand binding affinity and cell surface antigen density as key determinants of targeting efficiency. This study highlights how targeting small cell-surface molecules, such as the T antigen, can mediate a selective photodynamic treatment response which is similar to that achieved when targeting overexpressed protein receptors, such as HER-2. The high prevalence of the T antigen present on the cellular surface of primary tumours emphasises the broad potential applications for lectin-targeted therapies

Glycotope structures and intramolecular affinity factors of plant lectins for Tn/T antigens

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Structural basis for the carbohydrate specificities of artocarpin: variation in the length of a loop as a strategy for generating ligand specificity

Artocarpin, a tetrameric lectin of molecular mass 65 kDa, is one of the two lectins extracted from the seeds of jackfruit. The structures of the complexes of artocarpin with mannotriose and mannopentose reported here, together with the structures of artocarpin and its complex with Me-α-mannose reported earlier, show that the lectin possesses a deep-seated binding site formed by three loops. The binding site can be considered as composed of two subsites; the primary site and the secondary site. Interactions at the primary site composed of two of the loops involve mainly hydrogen bonds, while those at the secondary site comprising the third loop are primarily van der Waals in nature. Mannotriose in its complex with the lectin interacts through all the three mannopyranosyl residues; mannopentose interacts with the protein using at least three of the five mannose residues. The complexes provide a structural explanation for the carbohydrate specificities of artocarpin. A detailed comparison with the sugar complexes of heltuba, the only other mannose-specific jacalin-like lectin with known three-dimensional structure in sugar-bound form, establishes the role of the sugar-binding loop constituting the secondary site, in conferring different specificities at the oligosaccharide level. This loop is four residues longer in artocarpin than in heltuba, providing an instance where variation in loop length is used as a strategy for generating carbohydrate specificity

Verified and potential pathogens of predatory mites (Acari: Phytoseiidae)

Several species of phytoseiid mites (Acari: Phytoseiidae), including species of the genera Amblyseius, Galendromus, Metaseiulus, Neoseiulus, Phytoseiulus and Typhlodromus, are currently reared for biological control of various crop pests and/or as model organisms for the study of predator¿prey interactions. Pathogen-free phytoseiid mites are important to obtain high efficacy in biological pest control and to get reliable data in mite research, as pathogens may affect the performance of their host or alter their reproduction and behaviour. Potential and verified pathogens have been reported for phytoseiid mites during the past 25 years. The present review provides an overview, including potential pathogens with unknown host effects (17 reports), endosymbiotic Wolbachia (seven reports), other bacteria (including Cardinium and Spiroplasma) (four reports), cases of unidentified diseases (three reports) and cases of verified pathogens (six reports). From the latter group four reports refer to Microsporidia, one to a fungus and one to a bacterium. Only five entities have been studied in detail, including Wolbachia infecting seven predatory mite species, other endosymbiotic bacteria infecting Metaseiulus (Galendromus, Typhlodromus) occidentalis (Nesbitt), the bacterium Acaricomes phytoseiuli infecting Phytoseiulus persimilis Athias-Henriot, the microsporidium Microsporidium phytoseiuli infecting P. persimilis and the microsporidium Oligosproridium occidentalis infecting M. occidentalis. In four cases (Wolbachia, A. phytoseiuli, M. phytoseiuli and O. occidentalis) an infection may be connected with fitness costs of the host. Moreover, infection is not always readily visible as no obvious gross symptoms are present. Monitoring of these entities on a routine and continuous basis should therefore get more attention, especially in commercial mass-production. Special attention should be paid to field-collected mites before introduction into the laboratory or mass rearing, and to mites that are exchanged among rearing facilities. However, at present general pathogen monitoring is not yet practical as effects of many entities are unknown. More research effort is needed concerning verified and potential pathogens of commercially reared arthropods and those used as model organisms in research

Comparisons of host mitochondrial, nuclear and endosymbiont bacterial genes reveal cryptic fig wasp species and the effects of Wolbachia on host mtDNA evolution and diversity

Background Figs and fig-pollinating wasp species usually display a highly specific one-to-one association. However, more and more studies have revealed that the "one-to-one" rule has been broken. Co-pollinators have been reported, but we do not yet know how they evolve. They may evolve from insect speciation induced or facilitated by Wolbachia which can manipulate host reproduction and induce reproductive isolation. In addition, Wolbachia can affect host mitochondrial DNA evolution, because of the linkage between Wolbachia and associated mitochondrial haplotypes, and thus confound host phylogeny based on mtDNA. Previous research has shown that fig wasps have the highest incidence of Wolbachia infection in all insect taxa, and Wolbachia may have great influence on fig wasp biology. Therefore, we look forward to understanding the influence of Wolbachia on mitochondrial DNA evolution and speciation in fig wasps. Results We surveyed 76 pollinator wasp specimens from nine Ficus microcarpa trees each growing at a different location in Hainan and Fujian Provinces, China. We found that all wasps were morphologically identified as Eupristina verticillata, but diverged into three clades with 4.22-5.28% mtDNA divergence and 2.29-20.72% nuclear gene divergence. We also found very strong concordance between E. verticillata clades and Wolbachia infection status, and the predicted effects of Wolbachia on both mtDNA diversity and evolution by decreasing mitochondrial haplotypes. Conclusions Our study reveals that the pollinating wasp E. verticillata on F. microcarpa has diverged into three cryptic species, and Wolbachia may have a role in this divergence. The results also indicate that Wolbachia strains infecting E. verticillata have likely resulted in selective sweeps on host mitochondrial DNA

Method Development and Validation of Domperidone Malate in Tablet Dosage Form by Using RP-HPLC Method

A simple, precise, dependable, and replicable HPLC technique was developed to analyze Domperidone in solid dosage forms. The method utilized a C18 column with a mobile phase consisting of KH2PO4 Buffer and Methanol in a ratio of 60:40 (v/v), with detection performed at 253 nm. Domperidone exhibited a retention time of 6.7 minutes. Validation of the method was conducted following ICH guidelines, demonstrating satisfactory linearity, accuracy, and precision. The limit of detection and limit of quantification were determined to be 0.36 µg/mL and 1.56 µg/mL, respectively. This method proved suitable for the routine analysis of Domperidone, both individually and in combination with other dosage forms

Preserving Centromere Identity: Right Amounts of CENP-A at the Right Place and Time

Four decades ago, the discovery of centromere protein-A (CENP-A) marked a pivotal breakthrough in chromosome biology, revealing the epigenetic foundation of centromere identity. CENP-A, a histone H3 variant, directs the formation of the microtubule-binding kinetochore complex, designating the chromosomal site for its assembly and underpins the accurate partitioning of genetic material during cell division. Errors in cell division can give rise to DNA instability and aneuploidy, implicated in human diseases such as cancer. Therefore, discovering the underlying pathways and mechanisms responsible for the formation, regulation and maintenance of the centromere is important to our understanding of genome stability, epigenetic inheritance, and in providing the knowledge to help generate possible treatments and therapeutics. Here, we review various molecular pathways and mechanisms implicated in maintaining centromere identity and highlight some of the key outstanding questions with a focus on the human centromere

The Wolbachia Genome of Brugia malayi: Endosymbiont Evolution within a Human Pathogenic Nematode

Complete genome DNA sequence and analysis is presented for Wolbachia, the obligate alpha-proteobacterial endosymbiont required for fertility and survival of the human filarial parasitic nematode Brugia malayi. Although, quantitatively, the genome is even more degraded than those of closely related Rickettsia species, Wolbachia has retained more intact metabolic pathways. The ability to provide riboflavin, flavin adenine dinucleotide, heme, and nucleotides is likely to be Wolbachia's principal contribution to the mutualistic relationship, whereas the host nematode likely supplies amino acids required for Wolbachia growth. Genome comparison of the Wolbachia endosymbiont of B. malayi (wBm) with the Wolbachia endosymbiont of Drosophila melanogaster (wMel) shows that they share similar metabolic trends, although their genomes show a high degree of genome shuffling. In contrast to wMel, wBm contains no prophage and has a reduced level of repeated DNA. Both Wolbachia have lost a considerable number of membrane biogenesis genes that apparently make them unable to synthesize lipid A, the usual component of proteobacterial membranes. However, differences in their peptidoglycan structures may reflect the mutualistic lifestyle of wBm in contrast to the parasitic lifestyle of wMel. The smaller genome size of wBm, relative to wMel, may reflect the loss of genes required for infecting host cells and avoiding host defense systems. Analysis of this first sequenced endosymbiont genome from a filarial nematode provides insight into endosymbiont evolution and additionally provides new potential targets for elimination of cutaneous and lymphatic human filarial disease