Analysis of protein phosphorylation: methods and strategies for studying kinases and substrates

Protein phosphorylation is a highly conserved mechanism for regulating protein function, being found in all prokaryotes and eukaryotes examined. Phosphorylation can alter protein activity or subcellular localization, target proteins for degradation and effect dynamic changes in protein complexes. In many cases, different kinases may be involved in each of these processes for a single protein, allowing a large degree of combinatorial regulation at the post-translational level. Therefore, knowing which kinases are activated during a response and which proteins are substrates is integral to understanding the mechanistic regulation of a wide range of biological processes. In this paper, I will describe methods for monitoring kinase activity, investigating kinase-substrate specificity, examining phosphorylation in planta and the determination of phosphorylation sites in a protein. In addition, strategic considerations for experimental design and variables will be discussed.     

Intracellular Trafficking and Glycobiology of TbPDI2, a Stage-Specific Protein Disulfide Isomerase in Trypanosoma brucei

Trypanosoma brucei protein disulfide isomerase 2 (TbPDI2) is a bloodstream stage-specific lumenal endoplasmic reticulum (ER) glycoprotein. ER localization is dependent on the TbPDI2 C-terminal tetrapeptide (KQDL) and is mediated by TbERD2, an orthologue of the yeast ER retrieval receptor. Consistent with this function, TbERD2 localizes prominently to ER exit sites, and RNA interference (RNAi) knockdown results in specific secretion of a surrogate ER retention reporter, BiPN:KQDL. TbPDI2 is highly N-glycosylated and is reactive with tomato lectin, suggesting the presence of poly-N-acetyllactosamine modifications, which are common on lyso/endosomal proteins in trypanosomes but are inconsistent with ER localization. However, TbPDI2 is reactive with tomato lectin immediately following biosynthesis—far too rapidly for transport to the Golgi compartment, the site of poly-N-acetyllactosamine addition. TbPDI2 also fails to react with Erythrina cristagalli lectin, confirming the absence of terminal N-acetyllactosamine units. We propose that tomato lectin binds the Manβ1-4GlcNAcβ1-4GlcNAc trisaccharide core of paucimannose glycans on both newly synthesized and mature TbPDI2. Consistent with this proposal, α-mannosidase treatment renders oligomannose N-glycans on the T. brucei cathepsin L orthologue TbCatL reactive with tomato lectin. These findings resolve contradictory evidence on the location and glycobiology of TbPDI2 and provide a cautionary note on the use of tomato lectin as a poly-N-acetyllactosamine-specific reagent.     

Post-construction avian mortality monitoring at Project West Wind

Abstract

Post-construction avifauna investigations were undertaken at Project West Wind, Meridian Energy Limited's 62-turbine wind farm on the Wellington south coast. These investigations were required in accordance with the resource consent conditions to quantify the level of avian mortalities occurring at the wind farm, particularly in regard to New Zealand falcon (Falco novaeseelandiae), kākā (Nestor meridionalis) and kererū (Hemiphaga novaeseelandiae). This is the first comprehensive study at a New Zealand operating wind farm. The methods included three field components necessary to calculate annual estimates of mortalities across the wind farm site: routine turbine searches; carcass detection trials; and carcass removal trials. Results from years 1 and 2 of a three-year programme are presented. To date, mortalities have been recorded for 17 taxa at 18 of the 24 study turbines. There have been no recorded mortalities of falcon, kākā or kererū. Australasian harrier (Circus approximans) has been the species for which the most mortalities have been recorded. Overall estimated annual mortality rates for years 1 and 2 were calculated to be approximately six and five birds per turbine respectively.     

Sexual Dimorphism in Juvenile Hormone Synthesis by Corpora Allata and in Juvenile Hormone Acid Methyltransferase Activity in Corpora Allata and Accessory Sex Glands of Some Lepidoptera

Summary

The kinetic profiles of vitellin accumulation in the oyster ovary during oocyte growth and the effects in vivo and in vitro of estradiol-17β (E₂) on vitellin formation were examined in this study. The relative vitellin content measured by an enzyme-linked immunosorbent assay (ELISA) shows an apparent increase as the oocyte develops. Immunoblotting of the vitellin using anti-vitellin indicated that two main bands (179 and 110 kD), which begin to accumulate at an early stage of maturation, become pronounced during oocyte growth. Meanwhile, the major peak of the intact form of vitellin (530 kD) in gel filtration also enlarges with oocyte growth, supporting the results of immunoblot analysis and vitellin determination. E₂ treatment in vivo causes significant increases in oocyte diameter and vitellin content in the female oyster. A similar trend was observed in ovarian tissues cultured in the presence of E₂. It is concluded that E₂ is one of the major factors which control the vitellogenesis in the oyster and that the ovary is undoubtedly the site of synthesis of vitellin.     

Hooked on fat: the role of lipid synthesis in cancer metabolism and tumour development

An increased rate of lipid synthesis in cancerous tissues has long been recognised as an important aspect of the rewired metabolism of transformed cells. However, the contribution of lipids to cellular transformation, tumour development and tumour progression, as well as their potential role in facilitating the spread of cancerous cells to secondary sites, are not yet fully understood. In this article, we review the recent findings that support the importance of lipid synthesis and metabolism in tumorigenesis. Specifically, we explore the role of aberrant lipid biosynthesis in cancer cell migration and invasion, and in the induction of tumour angiogenesis. These processes are crucial for the dissemination of tumour cells and formation of metastases, which constitute the main cause of cancer mortality.     

The influence of probiotics on gastrointestinal tract infections among children attending childcare: A systematic review and meta-analysis

Current literature related to the impact of probiotics on the incidence of gastrointestinal tract infections (GITIs) has shown mixed results and no systematic review available with pooled analysis exists. Thus, the aim of this systematic review was to provide contemporary evidence regarding the overall and strain-specific influence of probiotics in preventing GITIs among infants and children attending childcare centres. The review shortlisted 18 RCTs after screening through the initial search results of 779 articles. However, only 15 trials were deemed eligible, addressing at least one outcome in the pooled analysis. It is concluded that the supplementation of probiotics (overall effect) may reduce the risk of GITI episode by 26%, with Lacticaseibacillus paracasei, Limosilactobacillus reuteri and Lacticaseibacillus rhamnosus GG being specifically potent probiotic strains in reducing GITI episode, duration of infection and absence from childcare respectively. There is insufficient evidence to determine the effect of Bifidobacterium animalis subsp. lactis BB-12 based on the findings of the trials included in this review.