Caenorhabditis elegans: a versatile platform for drug discovery

Drug discovery and drug target identification are two intimately linked facets of intervention strategies aimed at effectively combating pathological conditions in humans. Simple model organisms provide attractive platforms for devising and streamlining efficient drug discovery and drug target identification methodologies. The nematode worm Caenorhabditis elegans has emerged as a particularly convenient and versatile tool that can be exploited to achieve these goals. Although C. elegans is a relatively modern addition to the arsenal of model organisms, its biology has already been investigated to an exceptional level. This, coupled with effortless handling and a notable low cost of cultivation and maintenance, allows seamless implementation of high-throughput drug screening approaches as well as in-depth genetic and biochemical studies of the molecular pathways targeted by specific drugs. In this review, we introduce C. elegans as a model organism with significant advantages toward the identification of molecular drug targets. In addition, we discuss the value of the worm in the development of drug screening and drug evaluation protocols. The unique features of C. elegans, which greatly facilitate drug studies, hold promise for both deciphering disease pathogenesis and formulating educated and effective therapeutic interventions.     

Value of heartburn for diagnosing gastroesophageal reflux disease in severely obese patients

Objective : To evaluate the prevalence of gastroesophageal reflux disease (GERD) in severely obese patients and the association between symptoms and objective data of GERD in this population. Research Methods and Procedures : A total of 158 consecutive severely obese patients (BMI ≥ 40 kg/m²) were prospectively evaluated. Symptoms were evaluated by a structured clinical questionnaire. Objective assessment was made by ambulatory 24‐hour esophageal pH monitoring and endoscopy. GERD was defined by the presence of symptoms or complications (esophagitis). The clinical criterion defining GERD was the presence of at least two episodes of heartburn per week. Results : The mean age of the 138 patients subjected to complete study was 42.6 ± 10.2 years, with a BMI of 50.1 ± 6.9 kg/m² (range, 40.6 to 69.4 kg/m²); 78% were women. The prevalence of GERD evaluated by symptoms and/or esophagitis was 33.3% (46/138). Clinical criteria of GERD were present in 31/138 cases (22.5%), and 26 (18.8%) had esophagitis. In 69/138 patients (50%), pHmetry was abnormal. Fifty‐three patients with esophagitis and/or abnormal pHmetry were asymptomatic. The sensitivity of heartburn as a diagnostic criterion of GERD in patients with severe obesity was 29.3%, with a specificity of 85.7%. No significant association was observed between severe obesity grade and the prevalence of symptoms and/or objective data. Discussion : Asymptomatic gastroesophageal reflux (abnormal esophageal acid exposure and/or reflux esophagitis) is more common than symptomatic gastroesophageal reflux in severely obese patients. Increased BMI is not associated with a greater prevalence of GERD in these patients.     

Self-assembly of monoglycerides in beta-lactoglobulin adsorbed films at the air-water interface. Structural, topographical, and rheological consequences

In this work, we have analyzed the structural, topographical, and surface dilatational characteristics of pure beta-lactoglobulin adsorbed films and the effect of the self-assembly of monoglycerides (monopalmitin or monoolein) in beta-lactoglobulin films at the air-water interface. Measurements were performed in a single device that incorporates a Wilhelmy-type film balance, Brewster angle microscopy, and interfacial dilatational rheology. The structural and topographical characteristics of beta-lactoglobulin adsorbed and spread films are similar. However, the surface dilatational modulus of beta-lactoglobulin films shows a complex behavior depending on film formation. The self-assembly of monoglyceride in a beta-lactoglobulin adsorbed film has an effect on the structural, topographical, and dilatational properties of the mixed films, depending on the interfacial composition and the surface pressure (pi). At low pi, a mixed film of monoglyceride and beta-lactoglobulin may exist. At high pi (after the collapse of beta-lactoglobulin), the mixed films are dominated by monoglyceride molecules. However, the small amounts of collapsed beta-lactoglobulin have a significant effect on the surface dilatational properties of the mixed films. Protein displacement by monoglyceride is higher for monopalmitin than for monoolein. However, some degree of interaction exists between proteins and monoglycerides, and these interactions are more evident in adsorbed films than in spread films.     

Oral Administration of a GSK3 Inhibitor Increases Brain Insulin-like Growth Factor I Levels

Reduced brain input of serum insulin-like growth factor I (IGF-I), a potent neurotrophic peptide, may be associated with neurodegenerative processes. Thus, analysis of the mechanisms involved in passage of blood-borne IGF-I into the brain may shed light onto pathological mechanisms in neurodegeneration and provide new drug targets. A site of entrance of serum IGF-I into the brain is the choroid plexus. The transport mechanism for IGF-I in this specialized epithelium involves the IGF-I receptor and the membrane multicargo transporter megalin/LRP2. We have now analyzed this process in greater detail and found that the IGF-I receptor interacts with the transmembrane region of megalin, whereas the perimembrane domain of megalin is required for IGF-I internalization. Furthermore, a GSK3 site within the Src homology 3 domain of the C-terminal region of megalin is a key regulator of IGF-I transport. Thus, inhibition of GSK3 markedly increased internalization of IGF-I, whereas mutation of this GSK3 site abrogated this increase. Notably, oral administration of a GSK3 inhibitor to adult wild-type mice or to amyloid precursor protein/presenilin 1 mice modeling Alzheimer amyloidosis significantly increased brain IGF-I content. These results indicate that pharmacological modulation of IGF-I transport by megalin may be used to increase brain availability of serum IGF-I. Interestingly, GSK3 inhibitors such as those under development to treat Alzheimer disease may show therapeutic efficacy in part by increasing brain IGF-I levels, an effect already reported for other neuroprotective compounds.     

Oligomeric State in the Crystal Structure of Modular FAD Synthetase Provides Insights into Its Sequential Catalysis in Prokaryotes

The crystal structure of the modular flavin adenine dinucleotide (FAD) synthetase from Corynebacterium ammoniagenes has been solved at 1.95 Å resolution. The structure of C. ammoniagenes FAD synthetase presents two catalytic modules—a C-terminus with ATP-riboflavin kinase activity and an N-terminus with ATP-flavin mononucleotide (FMN) adenylyltransferase activity—that are responsible for the synthesis of FAD from riboflavin in two sequential steps. In the monomeric structure, the active sites from both modules are placed 40 Å away, preventing the direct transfer of the product from the first reaction (FMN) to the second catalytic site, where it acts as substrate. Crystallographic and biophysical studies revealed a hexameric assembly formed by the interaction of two trimers. Each trimer presents a head-tail configuration, with FMN adenylyltransferase and riboflavin kinase modules from different protomers approaching the active sites and allowing the direct transfer of FMN. Experimental results provide molecular-level evidences of the mechanism of the synthesis of FMN and FAD in prokaryotes in which the oligomeric state could be involved in the regulation of the catalytic efficiency of the modular enzyme.     

Species of the Paramecium aurelia complex in Italy

New stands of Paramecium pentaurelia were recorded in Valmarana, Veneto region in Italy and one stand of P. primaurelia was found at the same locality.     

IGF-I and insulin receptor signal transduction in trout muscle cells

In this study, primary cultures of trout skeletal muscle cells were used to investigate the main signal transduction pathways of insulin and IGF-I receptors in rainbow trout muscle. At different stages of in vitro development (myoblasts on day 1, myocytes on day 4, and fully developed myotubes on day 11), we detected in these cells the presence of immunoreactivity against ERK 1/2 MAPK and Akt/PKB proteins, components of the MAPK and the phosphatidylinositol 3-kinase-Akt pathways, respectively, two of the main intracellular transduction pathways for insulin and IGF-I receptors. Both insulin and IGF-I activated both pathways, although the latter provoked higher immunoreactivity of phosphorylated MAPKs and Akt proteins. At every stage, increases in total MAPK immunoreactivity levels were observed when cells were stimulated with IGF-I or insulin, while total Akt immunoreactivity levels changed little under stimulation of peptides. Total Akt and total MAPK levels increased as skeletal muscle cells differentiated in culture. Moreover, when cells were incubated with IGF-I or insulin, MAPK-P immunoreactivity levels showed greater increases over the basal levels on days 1 and 4, with no effect observed on day 11. Although Akt-P immunoreactivity displayed improved responses on days 1 and 4 as well, a stimulatory effect was still observed on day 11. In addition, the present study demonstrates that purified trout insulin receptors possess higher phosphorylative activity per unit of receptor than IGF-I receptors. In conclusion, these results indicate that trout skeletal muscle culture is a suitable model to study the insulin and IGF-I signal transduction molecules and that there is a different regulation of MAPK and Akt pathways depending on the developmental stage of the muscle cells.     

Antiproliferative effects of tocopherols (vitamin E) on murine glioma C6 cells: homologue-specific control of PKC/ERK and cyclin signaling

Chemoprevention strategies for brain tumors (specifically gliomas) are few and surprisingly poorly investigated. We have studied the effects of tocopherols (TOCs; vitamin E) on proliferation and death processes of murine glioma C6 cells. These vitamers showed different cell uptake and concentration- and time-dependent inhibitory effects on cell growth that were significant at the lowest concentrations tested (1-10 microM). However, the inhibitory potency of TOCs seemed to reflect at least in part their actual cell concentrations at steady state, with the order of magnitude gamma-TOC >or= alpha-TOC > delta-TOC approximately or = beta-TOC. Moreover, for extracellular concentrations >or=10 microM, TOCs also showed a significant cytotoxic effects due mainly to necrosis, while apoptosis was negligible. Gamma-TOC (the form showing preferential cell uptake and lowest unspecific cytotoxicity) was the most effective inhibitor of cell cycle progression (arrest in G0/G1 phase) leading to lowered expression of cyclin E and cyclin-dependent kinases 2 and 4 and overexpression of p27 (specific inhibitor of S-phase entering). According to these signals, activated ERK1/2 and PKC upstream and Rb phosphorylation downstream were decreased. In conclusion, within TOCs the gamma form exerts the most potent and specific control of cell cycle progression in C6 cells (cytostatic effect). This suggests a chemopreventive role of this form of vitamin E in gliomas.     

Oxidative DNA cleavage induced by an iron(III) flavonoid complex: synthesis, crystal structure and characterization of chlorobis(flavonolato)(methanol) iron(III) complex

A flavonol iron(III) complex, [Fe(flavonolato)(2)Cl(MeOH)], has been prepared. The compound has been characterized by X-ray crystallography, spectroscopy, magnetism and electronic paramagnetic resonance (EPR) at X- and Q-band. The geometrical environment around the metal is best described as rhombic distorted octahedral. This distortion has also been inferred from the magnetic measurements and from the EPR spectra at different temperatures, E/D(rhombicity parameter) approximately 0.06. The DNA cleavage activity of the iron(III) complex with and without ascorbate/hydrogen peroxide is reported. Mechanisms of the oxidative cleavage have been proposed when DNA strand scission is performed both with and without ascorbate/hydrogen peroxide activation.     

TaqMan PCR assay in the control of RNA normalization in human post-mortem brain tissue

The brain tissue obtained after death is subjected to several circumstances that can affect RNA integrity. The present study has been directed to reveal possible pitfalls and to control RNA normalization in post-mortem samples in order to recognize the limitations and minimize errors when using TaqMan PCR technology. This has been carried out in samples of the frontal cortex in a series of control and diseased cases covering Parkinson's disease, dementia with Lewy bodies pure form and common form, and Alzheimer's disease. Special attention has been paid to the value of the agonal state, post-mortem delay and pH of the nervous tissue as approximate predictors of the quality of RNA, as well as to the use of the Bioanalyzer to confirm RNA preservation. In addition, since possible disease-modified mRNAs have to be normalized with ideal unaltered RNAs, TaqMan human endogenous control plates have been used to determine the endogenous control most appropriate for the study. beta-glucuronidase (GUS) and beta-actin were good endogenous controls because their expression levels showed a small variation across a representative number of control and pathological cases. RNA stability was also analysed in a paradigm mimicking cumulative delay in tissue processing. GUS mRNA levels were not modified although beta-actin mRNA levels showed degradation at 22 h. Finally, the control of RNA degradation for the normalization of genes of interest was also tested. mRNA expression levels for superoxide dismutase 1 (SOD1) and metalloproteinase domain 22 (ADAM22) were examined at several artificial post-mortem times, and their expression levels compared with those for putative controls beta-actin and GUS. In our paradigm, the expressions of SOD1 and ADAM22 were apparently not modified when normalized with beta-actin. Yet their expression levels were reduced with post-mortem delay when values were normalized with GUS. Taken together, these observations point to practical consequences in TaqMan PCR studies. Short post-mortem delays and acceptable pH of the brain are not sufficient to rule out RNA degradation. The selection of adequate endogenous controls is pivotal in the study. beta-actin and GUS are found to be good endogenous controls in these pathologies, although GUS but not beta-actin expression levels are preserved in samples with long post-mortem delay.