Dealing with the genetic load in bacterial synthetic biology circuits: convergences with the Ohm's law.

Synthetic biology seeks to envision living cells as a matter of engineering. However, increasing evidence suggests that the genetic load imposed by the incorporation of synthetic devices in a living organism introduces a sort of unpredictability in the design process. As a result, individual part characterization is not enough to predict the behavior of designed circuits and thus, a costly trial-error process is eventually required. In this work, we provide a new theoretical framework for the predictive treatment of the genetic load. We mathematically and experimentally demonstrate that dependences among genes follow a quantitatively predictable behavior. Our theory predicts the observed reduction of the expression of a given synthetic gene when an extra genetic load is introduced in the circuit. The theory also explains that such dependence qualitatively differs when the extra load is added either by transcriptional or translational modifications. We finally show that the limitation of the cellular resources for gene expression leads to a mathematical formulation that converges to an expression analogous to the Ohm's law for electric circuits. Similitudes and divergences with this law are outlined. Our work provides a suitable framework with predictive character for the design process of complex genetic devices in synthetic biology.

Sex differences in the prognostic value of the lipid profile after the first ischemic stroke.

Post-stroke levels of total cholesterol (TC) appear to be negatively associated with stroke mortality. Statin pretreatment might affect this association. Sex differences in the prognostic value of the lipid profile have not yet been studied. We have evaluated the impact of TC, high- and low-density lipoprotein (HDL and LDL, respectively), and triglyceride (TG) levels on the 3-month outcome after a first ischemic stroke (IS) according to sex and previous statin use. The study group consisted of a hospital-based cohort of consecutive patients with a diagnosis of first IS. Poor outcome was defined as a modified Rankin Scale (mRS) score >or=3 at 90 days. The odds ration (OR) for poor prognosis was analyzed for each sex using logistic regression models adjusted for vascular risk factors and statin pretreatment. A total of 591 patients were included in the analysis (318 men). The predictors of a 90-day poor outcome were age and initial NIH Stroke Scale (NIHSS) score in women, and age, initial NIHSS, smoking, atrial fibrillation, and thrombolytic treatment in men. In women, none of the lipids studied affected the 90-day prognosis. Men falling in the last quintile of TC [OR: 0.68 95% confidence interval (95% CI) 0.52-0.88; p = 0.004] and LDL (OR 0.74, 95% CI 0.56-0.98; p = 0.04) have better outcome than men in the first quintile. Adjusting for statin pretreatment did not change the results. The results indicated that an association between poststroke lipids and prognosis may vary by sex. In women, lipids were not associated with the outcome; in men, lower TC and LDL were associated with worse prognosis. These differences can not be explained by statin use and require further research.

Angiotensin II induces contraction and proliferation of human hepatic stellate cells.

BACKGROUND & AIMS: Circulating levels of angiotensin II (ANGII), a powerful vasoconstrictor factor, are frequently increased in chronic liver diseases. In these conditions, hepatic stellate cells (HSCs) proliferate and acquire contractile properties. This study investigated the presence of receptors for ANGII and the effects of ANGII in human HSCs activated in culture. METHODS: The presence of ANGII receptors was assessed by binding studies. The effects of ANGII on intracellular calcium concentration ([Ca(2+)](i)), cell contraction, and cell proliferation were also assessed. RESULTS: Binding studies showed the presence of ANGII receptors of the AT1 subtype. ANGII elicited a marked dose-dependent increase in [Ca(2+)](i) and cell contraction. Moreover, ANGII stimulated DNA synthesis and increased cell number. All these effects were totally blocked by losartan and reduced by nitric oxide donors or prostaglandin E(2). The effects of ANGII were barely detectable in quiescent cells (2 days in culture), suggesting that phenotypic transformation of HSCs is associated with a marked increase in the effects of ANGII. CONCLUSIONS: ANGII induces contraction and is mitogenic for human-activated HSCs by acting through AT1 receptors. These results suggest that activated HSCs are targets of the vasoconstrictor action of ANGII in the intrahepatic circulation.

Human hepatic stellate cells secrete adrenomedullin: potential autocrine factor in the regulation of cell contractility.

BACKGROUND/AIMS: Hepatic stellate cells (HSCs) are perisinusoidal pericytes which have receptors for vasoactive factors, such as endothelin-1, which can regulate cell contractility in an autocrine manner. It is unknown whether human HSCs have receptors for and are able to synthesize the vasodilator peptide adrenomedullin (ADM), a peptide produced by most contractile cells. METHODS AND RESULTS: Stimulation of HSCs with ADM resulted in a dose-dependent raise in cAMP concentration (radioimmunoassay) and markedly blunted the endothelin-induced increase in [Ca2+]i and cell contraction, as assessed in cells loaded with fura-2 using a morphometric method. The existence of the receptor CRLR for ADM and their associated proteins RAMP-1 and RAMP-2 was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR). Moreover, activated human HSCs spontaneously secreted ADM in the culture medium in a time-dependent manner. ADM secretion was markedly enhanced by tumour necrosis factor-alpha and interleukin-1beta. Specific mRNA for ADM (RT-PCR and Northern blot) was detected in HSCs and increased after incubation of cells with cytokines. CONCLUSIONS: Human HSCs have functional receptors for ADM, the stimulation of which blunts the contractile effect of endothelin-1. Cultured human HSCs secrete ADM in baseline conditions. This secretion is markedly increased by cytokines. These results suggest that ADM can regulate HSCs' contractility in an autocrine manner.

Arginine vasopressin induces contraction and stimulates growth of cultured human hepatic stellate cells.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) are perisinusoidal cells believed to participate in the regulation of hepatic blood flow because of their contractile properties and presence of receptors for several vasoactive factors. It is unknown whether HSCs have receptors for vasopressin, one of the most potent endogenous vasoconstrictors. This study investigated the existence of receptors for and the effects of arginine vasopressin (AVP) on cultured human HSCs. METHODS: intracellular calcium concentration ([Ca2+]i) and cell contraction were measured in individual cells loaded with fura-2 using a morphometric method with an epifluorescence microscope coupled to a CCD imaging system (Photometrics, Tucson, AZ). AVP-specific binding was measured with [3H]AVP. Mitogen-activated protein kinase (MAPk) activity and DNA synthesis were measured by in vitro phosphorylation of myelin basic protein and [3H]thymidine incorporation, respectively. Parallel experiments were performed in vascular smooth muscle cells. RESULTS: AVP elicited a dose-dependent increase in [Ca2+]i and contraction of HSCs. Moreover, AVP increased MAPk activity, DNA synthesis, and cell number. These effects were similar to those observed in vascular smooth muscle cells and were blocked by a V1 receptor antagonist. The existence of V1 receptors was further confirmed by binding studies. CONCLUSIONS: Human HSCs have V1-vasopressin receptors that induce effects similar to those observed in vascular smooth muscle cells. AVP may play a role in the regulation of HSC function.

Contraction of human hepatic stellate cells activated in culture: a role for voltage-operated calcium channels.

BACKGROUND/AIMS: Voltage-operated calcium channels are essential for the regulation of vascular tone and are potential targets for vasodilating agents. They regulate calcium entry and thereby cell contraction in vascular cell types. Hepatic stellate cells in the activated phenotype have contractile properties and could participate in the regulation of sinusoidal blood flow. Thus, this study was aimed at investigating the presence of voltage-operated calcium channels in human hepatic stellate cells activated in culture and the effects of their stimulation on intracellular calcium concentration ([Ca2+]i) and cell contractility. METHODS: Binding studies using [3H]-nitrendipine were performed to demonstrate the presence of voltage-operated calcium channels. Voltage-operated calcium channels were stimulated by causing cell membrane depolarization either by electrical field stimulation or extracellular high potassium. [Ca2+]i and cell contraction were measured in individual cells loaded with fura-2 using a morphometric method with an epifluorescence microscope coupled to a charge-coupled device-imaging system. RESULTS: Binding studies demonstrated the existence of voltage-operated calcium channels in human activated hepatic stellate cells (7.1+/-1.4x10(4) sites/cell with a Kd of 2.1+/-0.1 nM). Both electrical field stimulation and potassium chloride-induced cell depolarization resulted in a marked and prolonged increase in [Ca2+]i followed by intense cell contraction. The degree of cell contraction correlated with the intensity of calcium peaks. Removal of extracellular calcium or preincubation of cells with nitrendipine, a specific antagonist of voltage-operated calcium channels, completely blocked the effects on [Ca2+]i and cell contraction, whereas preincubation of cells with BayK-8644, a specific agonist of voltage-operated calcium channels, increased calcium peaks and contraction. CONCLUSION: Activated human hepatic stellate cells have a large number of voltage-operated calcium channels, the activation of which is associated with an increase in [Ca2+]i followed by marked cell contraction. Voltage-operated calcium channels probably play an important role in the regulation of activated hepatic stellate cells contractility.

Atrial natriuretic peptide antagonizes endothelin-induced calcium increase and cell contraction in cultured human hepatic stellate cells.

Hepatic stellate cells (HSCs) participate in the regulation of hepatic microcirculation and have receptors for many vasoconstrictor factors. It is unknown whether HSCs have receptors for circulating vasodilators such as atrial natriuretic peptide (ANP). This study investigated the presence of ANP receptors in human HSCs and whether ANP antagonizes the effects of endothelin-1 in these cells. ANP receptors were assessed by binding and cross-linking studies, reverse-transcriptase polymerase chain reaction (PCR), and measuring intracellular cyclic guanosine monophosphate concentration. Intracellular calcium concentration ([Ca(2+)](i)) and cell contraction were measured in individual cells loaded with fura-2 using a morphometric method. Binding and cross-linking affinity experiments showed the existence of ANP receptors in human HSCs. PCR products with the expected length were obtained for guanylate cyclase A receptor, the physiological receptor of ANP, both in quiescent and activated human cells. ANP induced a dose-dependent increase in intracellular cyclic guanosine monophosphate concentration and blunted the increase in [Ca(2+)](i) elicited by endothelin-1. Most importantly, ANP markedly reduced cell contraction induced by endothelin-1. HSCs isolated from rats with carbon tetrachloride-induced cirrhosis showed a higher number of ANP receptors compared with HSCs isolated from normal rats, indicating that in vivo activation of HSCs is associated with an up-regulation of ANP receptors. These results indicate that human HSCs have receptors for ANP, the activation of which reduces the effects of endothelin-1 on [Ca(2+)](i) and cell contraction. ANP could participate in regulating the contractility of HSCs by antagonizing the effect of vasoconstrictors.