Sniffers,buzzers, toggles and blinkers: dynamics of regulatory and signaling pathways in the cell

The physiological responses of cells to external and internal stimuli are governed by genes and proteins interacting in complex networks whose dynamical properties are impossible to understand by intuitive reasoning alone. Recent advances by theoretical biologists have demonstrated that molecular regulatory networks can be accurately modeled in mathematical terms. These models shed light on the design principles of biological control systems and make predictions that have been verified experimentally.     

On the stall force for growing microtubules

The assembly of microtubules generates forces that play a role in cellular motility processes such as the motion of chromosomes during mitosis. Recently, Mogilner and Oster proposed a model for the growth of microtubules that agrees quantitatively with the force-velocity relation measured for individual microtubules. In addition, the authors predicted that the stall force for any polymer consisting of N independently growing protofilaments should increase as the square root of N. We simulated this model and found that the stall force increases linearly with N, and is in fact consistent with the maximum force predicted by thermodynamic arguments. We show that this discrepancy can be explained by a more careful treatment of the “off-term” in the Mogilner-Oster model. Received: 27 September 1999 / Revised version: 12 December 1999 / Accepted: 20 December 1999     

Bilayer undulation dynamics in unilamellar phospholipid vesicles: Effect of temperature,cholesterol and trehalose

We report a combined dynamic light scattering (DLS) and neutron spin-echo (NSE) study on the local bilayer undulation dynamics of phospholipid vesicles composed of 1,2-dimyristoyl-glycero-3-phosphatidylcholine (DMPC) under the influence of temperature and the additives cholesterol and trehalose. The additives affect vesicle size and self-diffusion. Mechanical properties of the membrane and corresponding bilayer undulations are tuned by changing lipid headgroup or acyl chain properties through temperature or composition. On the local length scale, changes at the lipid headgroup influence the bilayer bending rigidity κ less than changes at the lipid acyl chain: We observe a bilayer softening around the main phase transition temperature T_m of the single lipid system, and stiffening when more cholesterol is added, in concordance with literature. Surprisingly, no effect on the mechanical properties of the vesicles is observed upon the addition of trehalose.     

Potentiation of the glutamatergic synaptic input to rat locus coeruleus neurons by P2X7 receptors

Locus coeruleus (LC) neurons in a rat brain slice preparation were superfused with a Mg²⁺-free and bicuculline-containing external medium. Under these conditions, glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) were recorded by means of the whole-cell patch-clamp method. ATP, as well as its structural analogue 2-methylthio ATP (2-MeSATP), both caused transient inward currents, which were outlasted by an increase in the frequency but not the amplitude of the sEPSCs. PPADS, but not suramin or reactive blue 2 counteracted both effects of 2-MeSATP. By contrast, α,β-methylene ATP (α,β-meATP), UTP and BzATP did not cause an inward current response. Of these latter agonists, only BzATP slightly facilitated the sEPSC amplitude and strongly potentiated its frequency. PPADS and Brilliant Blue G, as well as fluorocitric acid and aminoadipic acid prevented the activity of BzATP. Furthermore, BzATP caused a similar facilitation of the miniature (m)EPSC (recorded in the presence of tetrodotoxin) and sEPSC frequencies (recorded in its absence). Eventually, capsaicin augmented the frequency of the sEPSCs in a capsazepine-, but not PPADS-antagonizable, manner. In conclusion, the stimulation of astrocytic P2X7 receptors appears to lead to the outflow of a signalling molecule, which presynaptically increases the spontaneous release of glutamate onto LC neurons from their afferent fibre tracts. It is suggested, that the two algogenic compounds ATP and capsaicin utilise separate receptor systems to potentiate the release of glutamate and in consequence to increase the excitability of LC neurons.     

Co‐ordinated autophagy with resveratrol and γ‐tocotrienol confers synergetic cardioprotection

This study compared two dietary phytochemicals, grape‐derived resveratrol and palm oil‐derived γ‐tocotrienol, either alone or in combination, on the contribution of autophagy in cardioprotection during ischaemia and reperfusion. Sprague‐Dawley rats weighing between 250 and 300 g were randomly assigned to one of the following groups: vehicle, ischaemia/reperfusion (I/R), resveratrol + I/R, γ‐tocotrienol + I/R, resveratrol +γ‐tocotrienol + I/R. For resveratrol treatments, the rats were gavaged with resveratrol (2.5 mg/kg) for 15 days while for γ‐tocotrienol experiments the rats were gavaged with γ‐tocotrienol (0.3 mg/kg) for 30 days. For the combined resveratrol +γ‐tocotrienol experiments, the rats were gavaged with γ‐tocotrienol for 15 days, and then gavaging continued with resveratrol along with γ‐tocotrienol for a further period of 15 days. After 30 days, isolated perfused hearts were subjected to 30 min. of global ischaemia followed by 2 hrs of reperfusion. Our results showed for the first time that at least in part, the cardioprotection (evidenced from the ventricular performance, myocardial infarct size and cardiomyocyte apoptosis) with resveratrol and γ‐toctrienol was achieved by their abilities to induce autophagy. Most importantly, resveratrol and γ‐tocotrienol acted synergistically providing greater degree of cardioprotection simultaneously generating greater amount of survival signal through the activation of Akt‐Bcl‐2 survival pathway. Autophagy was accompanied by the activation of Beclin and LC3‐II as well as mTOR signalling, which were inhibited by either 3‐methyl adenine (3‐MA) or Wortmannin. The autophagy was confirmed from the results of transmission electron microscopy and light microscopy as well as with confocal microscopy. It is tempting to speculate that during ischaemia and reperfusion autophagy along with enhanced survival signals helps to recover the cells from injury.     

Gender differences in biomechanical properties of intramural coronary resistance arteries of rats, an in vitro microarteriographic study

The prevalence of ischemic heart disease is lower in premenopausal females than in males of corresponding age. This should be related to gender differences in coronary functions. We tested whether biomechanical differences exist between intramural coronary resistance arteries of male and female rats. Intramural branches of the left anterior descending coronary artery (uniformly approximately 200microm in diameter) were isolated, cannulated and studied by microarteriography. Intraluminal pressure was increased from 2 to 90mmHg in steps and steady-state diameters were measured. Measurements were repeated in the presence of vasoconstrictor U46619 (10(-6)M) and the endothelial coronary vasodilator bradykinin (BK) (10(-6)M). Finally, passive diameters were recorded in calcium-free saline. A similar inner radius and a higher wall thickness (41.5+/-2.9microm vs. 31.4+/-2.7microm at 50mmHg in the passive condition, p<0.05) resulted in lower tangential wall stresses in male rats (18.9+/-1.9kPa vs. 24.9+/-2.5kPa at 50mmHg, p<0.05). Isobaric elastic modulus of vessels from male animals was significantly smaller at higher pressures. Vasoconstrictor response was significantly stronger in male than in female animals. Endothelial relaxations induced by BK were not different. This is the first demonstration that biomechanical characteristics of intramural coronary resistance arteries of a mammalian species are different in the male and female sexes. Higher wall thickness and higher vascular contractility in males are associated with similar endothelial function and larger high-pressure elasticity compared to females. These gender differences in biomechanics of coronary resistance arteries of rats may contribute to our better understanding the characteristic physiological and pathological differences in humans.     

Role of D327N sex hormone-binding globulin gene polymorphism in the pathogenesis of polycystic ovary syndrome

SHBG (sex hormone-binding globulin) is a transport protein specific for dihydrotestosterone, testosterone and estradiol. The missense mutation in exon 8 (GAC-->AAC) causing the amino acid exchange Asp-->Asn in codon 327 (D327N) correlates according to the published data with increased SHBG levels. We studied possible association of this polymorphism with polycystic ovary syndrome (PCOS) and anthropometric and biochemical parameters in 248 PCOS patients and 109 healthy control women. The D327N polymorphism (wild-type and variant allele) was detected using PCR-RFLP method (restriction enzyme Bbs-I). For statistical evaluation chi(2) test, Mann-Whitney test, ANCOVA, ANOVA (NCSS 2004, Statgraphics Plus v.5.1, USA) were used. There was no significant difference in genotype distribution between PCOS and controls (chi(2)=1.03, p=0.59). Moreover, we did not find an association of the variant allele with plasma SHBG level, steroid hormones, or screened parameters of lipid and glucose metabolism. In conclusion, the D327N polymorphism of the SHBG gene does not influence susceptibility to PCOS.     

Rapid detection of sex chromosomal aneuploidies by QF-PCR: application in 200 men with severe oligozoospermia or azoospermia

Klinefelter syndrome is the most common genetic cause of severe male factor infertility. Cytogenetic evaluation of metaphase chromosomes generally has a long turnaround time. We describe a reliable molecular genetic method that can be completed in 2 working days to identify the presence of any extra X chromosomes. The quantitative fluorescent (QF) 5-plex PCR includes the amplification of amelogenin, which is present on both sex chromosomes in a biallelic form, a polymorphic short tandem repeat (STR) on the pseudoautosomal region of X and Y (X22), two polymorphic X-specific STRs (DXS6803, DXS6809), and a Y-specific marker (SY134), in a single tube. The presence of an extra X chromosome is recognized either by a supernumerary peak or an increased peak area based on criteria we have developed. The application of the method on 200 patients resulted in the identification of 14 patients (7%) with Klinefelter syndrome or a variant form (2 SRY-positive 46,XX men), as well as an additional patient with 47,XYY karyotype. The QF-PCR method, along with Y chromosome microdeletion testing, can be used as a first-step genetic analysis in azoospermic or severely oligozoospermic patients for the rapid identification of sex chromosome aneuploidies.     

Spatial scaling of arbuscular mycorrhizal fungal diversity is affected by farming practice

Evidence suggests that microbial communities show patterns of spatial scaling which can be driven by geographical distance and environmental heterogeneity. Here we demonstrate that human management can have a major impact on microbial distribution patterns at both the local and landscape scale. Mycorrhizal fungi are vital components of terrestrial ecosystems, forming a mutualistic symbiosis with plant roots which has a major impact on above ground ecology and productivity. We used contrasting agricultural systems to investigate the spatial scaling of the most widespread mycorrhizal fungus group, the arbuscular mycorrhizal fungi (AMF). Using multiple sampling sites with a maximum separation of 250 km we describe for the first time the roles which land management, environmental heterogeneity and geographical distance play in determining spatial patterns of microbial distribution. Analysis of AMF taxa–area relationships at each sampling site revealed that AMF diversity and spatial turnover was greater under organic relative to conventional farm management. At the regional scale (250 km) distance–decay analyses showed that there was significant change in AMF community composition with distance, and that this was greater under organic relative to conventional management. Environmental heterogeneity was found to be the major factor determining turnover of AMF taxa at the landscape scale. Overall we demonstrate that human management can play a key role in determining the turnover of microbial communities at both the local and regional scales.     

Grp78, Grp94, and Grp170 interact with alpha1-antitrypsin mutants that are retained in the endoplasmic reticulum

In alpha1-antitrypsin (alpha1-AT) deficiency, a mutant form of alpha1-AT polymerizes in the endoplasmic reticulum (ER) of liver cells resulting in chronic hepatitis and hepatocellular carcinoma by a gain of toxic function mechanism. Although some aspects of the cellular response to mutant alpha1-AT Z have been partially characterized, including the involvement of several proteasomal and nonproteasomal mechanisms for disposal, other parts of the cellular response pathways, particularly the chaperones with which it interacts and the signal transduction pathways that are activated, are still not completely elucidated. The alpha1-AT Z molecule is known to interact with calnexin, but, according to one study, it does not interact with Grp78. To carry out a systematic search for the chaperones with which alpha1-AT Z interacts in the ER, we used chemical cross-linking of several different genetically engineered cell systems. Mutant alpha1-AT Z was cross-linked with Grp78, Grp94, calnexin, Grp170, UDP-glucose glycoprotein:glucosyltransferase, and two unknown proteins of approximately 110-130 kDa. Sequential immunoprecipitation/immunoblot analysis and coimmunoprecipitation techniques demonstrated each of these interactions without chemical cross-linking. The same chaperones were found to interact with two nonpolymerogenic alpha1-AT mutants that are retained in the ER, indicating that these interactions are not specific for the alpha1-AT Z mutant. Moreover, sucrose density gradient centrifugation studies suggest that approximately 85% of alpha1-AT Z exists in heterogeneous soluble complexes with multiple chaperones and approximately 15% in extremely large polymers/aggregates devoid of chaperones. Agents that perturb the synthesis and/or activity of ER chaperones such as tunicamycin and calcium ionophore A23187, have different effects on the solubility and degradation of alpha1-AT Z as well as on its residual secretion.