Bioactivity of glycogen phosphorylase inhibitors that bind to the purine nucleoside site

The bioactivity in hepatocytes of glycogen phosphorylase inhibitors that bind to the active site, the allosteric activator site and the indole carboxamide site has been described. However, the pharmacological potential of the purine nucleoside inhibitor site has remained unexplored. We report the chemical synthesis and bioactivity in hepatocytes of four new olefin derivatives of flavopiridol (1-4) that bind to the purine site. Flavopiridol and 1-4 counteracted the activation of phosphorylase in hepatocytes caused by AICAR (5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside), which is metabolised to an AMP analogue. Unlike an indole carboxamide inhibitor, the analogues 1 and 4 suppressed the basal rate of glycogenolysis in hepatocytes by allosteric inhibition rather than by inactivation of phosphorylase, and accordingly caused negligible stimulation of glycogen synthesis. However, they counteracted the stimulation of glycogenolysis by dibutyryl cAMP by both allosteric inhibition and inactivation of phosphorylase. Cumulatively, the results show key differences between purine site and indole carboxamide site inhibitors in terms of (i) relative roles of dephosphorylation of phosphorylase-a as compared with allosteric inhibition, (ii) counteraction of the efficacy of the inhibitors on glycogenolysis by dibutyryl-cAMP and (iii) stimulation of glycogen synthesis.     

Reduction of all-trans-retinal in vertebrate rod photoreceptors requires the combined action of RDH8 and RDH12

In vertebrate rod cells, retinoid dehydrogenases/reductases (RDHs) are critical for reducing the reactive aldehyde all-trans-retinal that is released by photoactivated rhodopsin, to all-trans-retinol (vitamin A). Previous studies have shown that RDH8 localizes to photoreceptor outer segments and is a strong candidate for performing this role. However, RDH12 function in the photoreceptor inner segments is also key, because loss of function mutations cause retinal degeneration in some forms of Leber congenital amaurosis. To investigate the in vivo roles of RDH8 and RDH12, we used fluorescence imaging to examine all-trans-retinol production in single isolated rod cells from wild-type mice and knock-out mice lacking either one or both RDHs. Outer segments of rods deficient in Rdh8 failed to reduce all-trans-retinal, but those deficient in Rdh12 were unaffected. Following exposure to light, a leak of retinoids from outer to inner segments was detected in rods from both wild-type and knock-out mice. In cells lacking Rdh8 or Rdh12, this leak was mainly all-trans-retinal. Wild-type rods incubated with all-trans-retinal reduced moderate loads of retinal within the cell interior, but this ability was lost by cells deficient in Rdh8 or Rdh12. Our findings are consistent with localization of RDH8 to the outer segment where it provides most of the activity needed to reduce all-trans-retinal generated by the light response. In contrast, RDH12 in inner segments can protect vital cell organelles against aldehyde toxicity caused by an intracellular leak of all-trans-retinal, as well as other aldehydes originating both inside and outside the cell.     

Gene regulatory networks from multifactorial perturbations using Graphical Lasso: application to the DREAM4 challenge

A major challenge in the field of systems biology consists of predicting gene regulatory networks based on different training data. Within the DREAM4 initiative, we took part in the multifactorial sub-challenge that aimed to predict gene regulatory networks of size 100 from training data consisting of steady-state levels obtained after applying multifactorial perturbations to the original in silico network. Due to the static character of the challenge data, we tackled the problem via a sparse Gaussian Markov Random Field, which relates network topology with the covariance inverse generated by the gene measurements. As for the computations, we used the Graphical Lasso algorithm which provided a large range of candidate network topologies. The main task was to select the optimal network topology and for that, different model selection criteria were explored. The selected networks were compared with the golden standards and the results ranked using the scoring metrics applied in the challenge, giving a better insight in our submission and the way to improve it.Our approach provides an easy statistical and computational framework to infer gene regulatory networks that is suitable for large networks, even if the number of the observations (perturbations) is greater than the number of variables (genes).     

Walking kinematics and kinetics following eccentric exercise-induced muscle damage

The goal of this investigation was to investigate how walking patterns are affected following muscle-damaging exercise by quantifying both lower limb kinematics and kinetics. Fifteen young women conducted a maximal isokinetic eccentric exercise (EE) muscle damage protocol (5 × 15) of the knee extensors and flexors of both legs at 60°/s. Three-dimensional motion data and ground reaction forces (GRFs) were collected 24 h pre-EE while the participants walked at their preferred self-selected walking speed (SWS). Participants were asked to perform two gait conditions 48 h post-EE. The first condition (COND1) was to walk at their own speed and the second condition (COND2) to maintain the SWS (±5%) they had 24 h pre-EE. Walking speed during COND1 was significantly lower compared to pre-exercise values. When walking speed was controlled during COND2, significant effects of muscle damage were noticed, among other variables, for stride frequency, loading rate, lateral and vertical GRFs, as well as for specific knee kinematics and kinetics. These findings provide new insights into how walking patterns are adapted to compensate for the impaired function of the knee musculature following muscle damage. The importance to distinguish the findings caused by muscle damage from those exhibited in response to changes in stride frequency is highlighted.     

Increased cellular apoptosis after chronic aqueous exposure to nonylphenol and quercetin in adult medaka (Oryzias latipes)

Increasing evidence suggests that sublethal effects of natural or xenobiotic chemicals in the environment may be mediated via the stimulation of apoptosis. To investigate whether apoptosis can be induced in fish by weakly estrogenic and androgenic chemicals, adult male Japanese medaka (Oryzias latipes) were exposed to 100 ppb of the estrogenic alkylphenol, 4-nonylphenol, and adult female medaka were exposed to 100 ppb of the aromatase-inhibiting bioflavonoid, quercetin, for 6 weeks. Exposure to nonylphenol and quercetin had no significant effect on the length, weight or condition factors compared to solvent (acetone) controls in male or female medaka. Apoptosis was evaluated in blinded histological sections of whole medaka using terminal dideoxynucleotidyl transferase dUTP nick end labeling (TUNEL) that labels nuclei of cells containing apoptotic (fragmented) DNA. There was a six-fold greater extent of apoptosis in spermatocytes, Sertoli cells and Leydig-homologue cells, but not in spermatids of testes from nonylphenol-exposed male medaka compared to testes of solvent controls. No significant differences in the extent of apoptosis were detected in intestine, liver or kidney from the same male fish. Quercetin-treated female medaka had a significantly increased number of atretic ovarian follicles, but no significant differences in the extent of apoptosis in intestine, liver or kidney. These results suggest that nonylphenol caused testicular degeneration via increased testicular cell apoptosis, while quercetin may be ovotoxic via increased follicular atresia.     

A proteomic approach to investigate the differential antigenic profile of two Coxiella burnetii strains

Q fever is a widespread zoonosis caused by Coxiella burnetii, an obligate intracellular Gram-negative bacterium. Current diagnostics of Q fever is based on serological testing of patient serum. Biological distinction among C. burnetii strains has been referred at the genetic level as well as in virulence in animal models of Q fever. Disclosure of strain specific antigens might show insight into the biology and pathogenesis of this query pathogen, as well as it can provide the literature with potential serodiagnostic markers. In the present study, we sought to obtain an outer membrane enriched fraction of two C. burnetii reference strains, which originate from different sources, in order to investigate the way in which their antigenic profile is differentiated against a patient serum. We systematically analyzed the sarcosyl-insoluble fraction, enriched in outer membrane proteins, of the two C. burnetii strains using doubled SDS-PAGE combined with MS/MS analysis. In total, twenty-two outer membrane proteins were identified, representing 26% of the overall 86 identified proteins. The sarcosyl-insoluble fraction was then separated on 2DE IEF/SDS-PAGE and probed with serum from an infected patient. Different immuno-reactive proteins between the two C. burnetii strains were identified and included 2 outer membrane proteins, a hypothetical protein (CBU_0937) with unknown function and OmpH (CBU_0612), a previously identified marker for Q fever endocarditis. This approach can be used to reveal strain-specific proteins involved in pathogenesis and new serodiagnostic markers.     

Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells^1-4. Rod photoreceptors are responsible for vision in dim light, cones in bright light. Phototransduction takes place in the outer segment of the photoreceptor cell, a specialized compartment that contains a high concentration of visual pigment, the primary light detector. The visual pigment is composed of a chromophore, 11-cis retinal, attached to a protein, opsin. A photon absorbed by the visual pigment isomerizes the chromophore from 11-cis to all-trans. This photoisomerization brings about a conformational change in the visual pigment that initiates a cascade of reactions culminating in a change in membrane potential, and bringing about the transduction of the light stimulus to an electrical signal. The recovery of the cell from light stimulation involves the deactivation of the intermediates activated by light, and the reestablishment of the membrane potential. Ca²⁺ modulates the activity of several of the enzymes involved in phototransduction, and its concentration is reduced upon light stimulation. In this way, Ca²⁺ plays an important role in the recovery of the cell from light stimulation and its adaptation to background light.Another essential part of the recovery process is the regeneration of the visual pigment that has been destroyed during light-detection by the photoisomerization of its 11-cis chromophore to all-trans^5-7. This regeneration begins with the release of all-trans retinal by the photoactivated pigment, leaving behind the apo-protein opsin. The released all-trans retinal is rapidly reduced in a reaction utilizing NADPH to all- trans retinol, and opsin combines with fresh 11-cis retinal brought into the outer segment to reform the visual pigment. All-trans retinol is then transferred out of the outer segment and into neighboring cells by the specialized carrier Interphotoreceptor Retinoid Binding Protein (IRBP).Fluorescence imaging of single photoreceptor cells can be used to study their physiology and cell biology. Ca²⁺-sensitive fluorescent dyes can be used to examine in detail the interplay between outer segment Ca²⁺ changes and response to light^8-12 as well as the role of inner segment Ca²⁺ stores in Ca²⁺ homeostasis^13,14. Fluorescent dyes can also be used for measuring Mg²⁺ concentration¹⁵, pH, and as tracers of aqueous and membrane compartments¹⁶. Finally, the intrinsic fluorescence of all-trans retinol (vitamin A) can be used to monitor the kinetics of its formation and removal in single photoreceptor cells^17-19.Download video file.^{(70M, mov)}     

Stochastic simulations of the tetracycline operon

Background  

The tetracycline operon is a self-regulated system. It is found naturally in bacteria where it confers resistance to antibiotic tetracycline. Because of the performance of the molecular elements of the tetracycline operon, these elements are widely used as parts of synthetic gene networks where the protein production can be efficiently turned on and off in response to the presence or the absence of tetracycline. In this paper, we investigate the dynamics of the tetracycline operon. To this end, we develop a mathematical model guided by experimental findings. Our model consists of biochemical reactions that capture the biomolecular interactions of this intriguing system. Having in mind that small biological systems are subjects to stochasticity, we use a stochastic algorithm to simulate the tetracycline operon behavior. A sensitivity analysis of two critical parameters embodied this system is also performed providing a useful understanding of the function of this system.