Exploring the Intracrine Functions of VEGF-A

Vascular endothelial growth factor A (VEGF-A or VEGF) is a highly conserved secreted signalling protein best known for its roles in vascular development and angiogenesis. Many non-endothelial roles for VEGF are now established, with the discovery that VEGF and its receptors VEGFR1 and VEGFR2 are expressed in many non-vascular cell-types, as well as various cancers. In addition to secreted VEGF binding to its receptors in the extracellular space at the cell membrane (i.e., in a paracrine or autocrine mode), intracellularly localised VEGF is emerging as an important signalling molecule regulating cell growth, survival, and metabolism. This intracellular mode of signalling has been termed “intracrine”, and refers to the direct action of a signalling molecule within the cell without being secreted. In this review, we describe examples of intracrine VEGF signalling in regulating cell growth, differentiation and survival, both in normal cell homeostasis and development, as well as in cancer. We further discuss emerging evidence for the molecular mechanisms underpinning VEGF intracrine function, as well as the implications this intracellular mode of VEGF signalling may have for use and design of anti-VEGF cancer therapeutics.

Cysteine immobilisation on the polyethylene terephthalate surfaces and its effect on the haemocompatibility

Nitric oxide (NO) is an important signalling molecule involved in haemostasis. NO, present as endogenous S-nitrosothiols, is released by cysteine through a transnitrosation reaction. To exploit this mechanism, cysteine was immobilised onto the different carboxylated polyethylene terephthalate (PET) surfaces using 1-step EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) crosslinking mechanism. Immobilised cysteine concentration and NO release were dependent on the surface carboxyl density. Stability studies showed that the immobilised cysteine concentration and NO release reduced within 6 h. Immobilisation of cysteine derivatives eliminated the possibility of formation of polycysteine and its electrostatic interaction with the carboxylated PET. The immobilised cysteine concentration did not recover after DTT treatment, eliminating the possibility of disulphide bond formation. Further, cysteine was immobilised using a 2-step EDC crosslinking mechanism. Although the cysteine concentration reduced during stability studies, it recovered upon DTT treatment, indicating that cysteine forms amide bonds with the carboxylated PET and the observed decrease in cysteine concentration is probably due to the formation of disulphide bonds. The haemocompatibility of the cysteine immobilised PET surfaces showed similar results compared to the carboxylated PET. The loss of thiol groups due to the disulphide bond restricts the transnitrosation reaction. Hence, these materials can be used primarily in short-term applications.

Current approaches to measure nitric oxide in plants

Nitric oxide (NO) is now established as an important signalling molecule in plants where it influences growth, development, and responses to stress. Despite extensive research, the most appropriate methods to measure and localize these signalling radicals are debated and still need investigation. Many confounding factors such as the presence of other reactive intermediates, scavenging enzymes, and compartmentation influence how accurately each can be measured. Further, these signalling radicals have short half-lives ranging from seconds to minutes based on the cellular redox condition. Hence, it is necessary to use sensitive and specific methods in order to understand the contribution of each signalling molecule to various biological processes. In this review, we summarize the current knowledge on NO measurement in plant samples, via various methods. We also discuss advantages, limitations, and wider applications of each method.

Keeping the balance in NAD metabolism

Research over the last few decades has extended our understanding of nicotinamide adenine dinucleotide (NAD) from a vital redox carrier to an important signalling molecule that is involved in the regulation of a multitude of fundamental cellular processes. This includes DNA repair, cell cycle regulation, gene expression and calcium signalling, in which NAD is a substrate for several families of regulatory proteins, such as sirtuins and ADP-ribosyltransferases. At the molecular level, NAD-dependent signalling events differ from hydride transfer by cleavage of the dinucleotide into an ADP-ribosyl moiety and nicotinamide. Therefore, non-redox functions of NAD require continuous biosynthesis of the dinucleotide. Maintenance of cellular NAD levels is mainly achieved by nicotinamide salvage, yet a variety of other precursors can be used to sustain cellular NAD levels via different biosynthetic routes. Biosynthesis and consumption of NAD are compartmentalised at the subcellular level, and currently little is known about the generation and role of some of these subcellular NAD pools. Impaired biosynthesis or increased NAD consumption is deleterious and associated with ageing and several pathologies. Insults to neurons lead to depletion of axonal NAD and rapid degeneration, partial rescue can be achieved pharmacologically by administration of specific NAD precursors. Restoring NAD levels by stimulating biosynthesis or through supplementation with precursors also produces beneficial therapeutic effects in several disease models. In this review, we will briefly discuss the most recent achievements and the challenges ahead in this diverse research field.

Synthesis of an inositol hexakisphosphate (IP6) affinity probe to study the interactome from a colon cancer cell line

Inositol hexakisphosphate (InsP6or IP6) is an important signalling molecule. An IP6probe was synthesised frommyo-inositol and immobilised onto Dynabeads for proteomic analysis. LC/MS/MS analysis identified 77 proteins or protein complexes that bind to IP6specifically.