Current Chemical Biology - Volume 7, Issue 3, 2013

We have used the Smoldyn software to create spatially detailed models of the Escherichia coli chemotaxis pathway. With it we can follow signalling reactions at high spatio-temporal resolution, observe the formation of gradients of phospho-proteins as well as total protein and analyse the effects of macromolecular crowding on signalling. It has enabled us to propose new regulatory elements of the signalling pathway, which are mediated through the dynamic localisation and activity of the CheZ phosphatase. We used the Smoldyn software to model quantitative fluorescent microscopy data and to determine diffusion coefficients and binding affinities. We can reliably gain information obtained under conditions with high levels of experimental noise. Smoldyn can accommodate various cellular architectures, and we show that cell shape becomes an integral part of the signalling process.

In this review we discuss the interaction between experiments and theory in understanding regulatory systems in bacterial cells, using the galactose network of E. coli as a model system to illustrate this. Initially, experiments led to a coarse-grained network view of this system, which revealed the structure of entangled negative and positive feedback loops that regulates galactose metabolism. A dynamical model, based on this network picture and in vitro experiments, indicated that one of the main regulators would have little role to play in any steady-state scenarios. The dynamical experiments this result suggested revealed the intricate timing sequence of gene expression in situations of rapidly decreasing galactose. All this information is slowly coming together to form a picture of how the different elements of the galactose regulatory system work together to regulate metabolism in the presence of fluctuating and changing food sources.

Proteins interact with each other to perform an array of cellular functions. A systems-level knowledge of protein interaction networks, as well as the underlying domain-domain interactions, constitutes a major goal in systems biology. In this mini-review, we discuss some background to the problem of inference of domain-domain interactions and briefly discuss some relevant computational and statistical approaches. Specifically, we review our earlier work in which we formulate this problem using a graphical model combined with inference of domain-interaction probabilities using powerful statistical inference methods, namely belief propagation. The inferred interactions can be used for analysing network data, and, by looking at the specificity of protein-protein interactions, they can also be used to predict novel protein interactions within and across orthologous species.

Snake venom contains a number of different, pharmacologically-active proteins and peptides. Most of the haemorrhagic proteins of snake venoms are metalloproteinases. Agkistrodon halys metalloproteinase (AHM) was isolated from the snake venom of Pallas (Mol wt. 23145). In vitro toxicological effects of AHM (0.1-2 mM) on human macrophages (THP-1 and U-937), lung fibroblasts (MRC-5) and murine lung epithelial (LA4) cells were evaluated by (2,3-bis- (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-hydroxide) assay and light microscopy. AHM strongly inhibited cell proliferation and adhesion to extracellular matrix, as well as induced morphological changes in a dose-dependent manner. Apoptosis was evaluated using propidium iodide (PI) staining and a terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay for DNA fragmentation. PI staining indicated an accumulation of cells at the sub-G1-phase following AHM treatment, and there was also DNA fragmentation as shown by TUNEL staining. Besides cell-based assays, an in vivo assessment of AHM (1.56-300 mg/kg, body weight) in mice was also done. Histopathology of muscle fibers revealed massive necrotic aggregations after AHM exposure. There were translucent vacuoles in the purkinje cells, which may cause substantial damage to kidney tubular epithelium. There were also clear areas in the cerebellum due to cell death, deposition of fibrinogen or fibrin on the intestinal epithelium, and skin necrosis following an AHM dose of 300 mg/kg. We also observed marked erythrocyte accumulation in lung alveolar walls that resulted in infarction, along with a consequent reduction of the alveolar space and necrosis linked to neutrophil infiltration. These results cumulatively suggest that AHM induces lethality at high doses, inhibits cell proliferation, and induces morphological changes in various cell types.

Mainly synthesized in the stomach, ghrelin is a peptide hormone which stimulates growth hormone secretion and appetite, thus promoting food intake and body-weight gain. Historically, researchers started to work on the discovery of ghrelin receptor ligands several years before the discovery of the ghrelin receptor and the hormone itself. Indeed peptides able to stimulate growth hormone secretion (growth hormone releasing peptides, GHRPs) were found while the mechanism of action and the target receptor were still unknown. Non peptidic agonists were then described (growth hormone secretagogues, GHSs) and the receptor (GHS-R1a) identified in 1996. Three years later, the natural ligand of this receptor (ghrelin) was isolated from stomach and its chemical synthesis allowed to show the physiological role of ghrelin in energy balance. In this review, we present some pseudopeptide and peptidomimetic approaches used by researchers for the design of ghrelin receptor ligands. We will start by the pioneering work of Bowers et al. on enkephalin analogues, which was the starting point for the development of an impressive number of compounds, by several of the major worldwide pharma companies. We will also describe the work achieved starting from a substance P derivative, which was one of the first peptides identified as an antagonist of the newly discovered ghrelin receptor. Then we will review the structure activity relationship study starting from the peptide ghrelin, which started with the discovery of this peptide in 1999. We will also focus on a more recent work based on macrocyclic peptidic analogues for the development of ghrelin receptor ligands.

There is a vast literature dealing with various aspects of the circadian clock. This review provides examples of the multifaceted nature, of namely redox, oxidative stress, melatonin, antioxidants, nitric oxide, hypoxia, anesthetics, cortisol and cocaine. The redox and oxidative stress features reflect the involvement of electron transfer and radical species, which can exert either beneficial or deleterious effects. The harmful aspects can be countered by antioxidants. Melatonin is a brain hormone that is known to play a widespread role in the biochemistry of the circadian clock. Nitric oxide is widespread in the human body and plays a variety of roles. Hypoxia has been broadly discussed in relation to circadian rhythm, e.g., in high altitude flight. The anesthetic topic completes the list.

Sertoli cells (SCs) are essential for the testis functional development and hence for the expression of male phenotype. They provide a unique and protected environment in testis, within the seminiferous tubules, necessary for the successful progression of germ cells into fully competent spermatozoa. SC has the ability to metabolize various substrates, but preferentially uses glucose that is converted to pyruvate and lactate. This is a crucial event since lactate is essential for germ cells survival and development due to its anti-apoptotic effect and its role as energy source. Glucose metabolism in SCs is under the complex control of several hormones, predominantly sex steroid hormones, thyroid hormones (THs): follicle- stimulating hormone (FSH) and insulin. This process may occur without the requirement of protein synthesis, suggesting a modulation of enzyme activity and/or regulation of glucose transport. The transport of glucose through the plasma membrane contributes to the modulation of lactate secretion by SCs and is mediated by specific carriers. There are two different families of glucose transport proteins: the Sodium Dependent Glucose Transporters (SGLTs) and the Glucose Transporters (GLUTs): which act in a very distinct manner. The maintenance of spermatogenesis in vivo and the male fertility capacity requires a metabolic cooperation between SCs and germ cells. Indeed, an alteration in SCs ability to metabolize glucose would be expected to compromise the energy supplies to germ cells and subsequently male fertility. Herein, we discuss the regulatory molecular mechanisms of glucose transport and metabolism in SC as well as their relevance for male fertility.