S-Leaping: An Adaptive, Accelerated Stochastic Simulation Algorithm, Bridging [Formula: see text]-Leaping and R-Leaping.

We propose the S-leaping algorithm for the acceleration of Gillespie's stochastic simulation algorithm that combines the advantages of the two main accelerated methods; the [Formula: see text]-leaping and R-leaping algorithms. These algorithms are known to be efficient under different conditions; the [Formula: see text]-leaping is efficient for non-stiff systems or systems with partial equilibrium, while the R-leaping performs better in stiff system thanks to an efficient sampling procedure. However, even a small change in a system's set up can critically affect the nature of the simulated system and thus reduce the efficiency of an accelerated algorithm. The proposed algorithm combines the efficient time step selection from the [Formula: see text]-leaping with the effective sampling procedure from the R-leaping algorithm. The S-leaping is shown to maintain its efficiency under different conditions and in the case of large and stiff systems or systems with fast dynamics, the S-leaping outperforms both methods. We demonstrate the performance and the accuracy of the S-leaping in comparison with the [Formula: see text]-leaping and R-leaping on a number of benchmark systems involving biological reaction networks.

Cost-benefit tradeoffs in engineered lac operons.

Cells must balance the cost and benefit of protein expression to optimize organismal fitness. The lac operon of the bacterium Escherichia coli has been a model for quantifying the physiological impact of costly protein production and for elucidating the resulting regulatory mechanisms. We report quantitative fitness measurements in 27 redesigned operons that suggested that protein production is not the primary origin of fitness costs. Instead, we discovered that the lac permease activity, which relates linearly to cost, is the major physiological burden to the cell. These findings explain control points in the lac operon that minimize the cost of lac permease activity, not protein expression. Characterizing similar relationships in other systems will be important to map the impact of cost/benefit tradeoffs on cell physiology and regulation.

Echocardiographic assessment of cardiac function in diabetic db/db and transgenic db/db-hGLUT4 mice.

Control db/+ and diabetic db/db mice at 6 and 12 wk of age were subjected to echocardiography to determine whether contractile function was reduced in vivo and restored in transgenic db/db-human glucose transporter 4 (hGLUT4) mice (12 wk old) in which cardiac metabolism has been normalized. Systolic function was unchanged in 6-wk-old db/db mice, but fractional shortening and velocity of circumferential fiber shortening were reduced in 12-wk-old db/db mice (43.8 +/- 2.1% and 8.3 +/- 0.5 circs/s, respectively) relative to db/+ control mice (59.5 +/- 2.3% and 11.8 +/- 0.4 circs/s, respectively). Doppler flow measurements were unchanged in 6-wk-old db/db mice. The ratio of E and A transmitral flows was reduced from 3.56 +/- 0.29 in db/+ mice to 2.40 +/- 0.20 in 12-wk-old db/db mice, indicating diastolic dysfunction. Thus a diabetic cardiomyopathy with systolic and diastolic dysfunction was evident in 12-wk-old diabetic db/db mice. Cardiac function was normalized in transgenic db/db-hGLUT4 mice, indicating that altered cardiac metabolism can produce contractile dysfunction in diabetic db/db hearts.

Rainbow trout glucose transporter (OnmyGLUT1): functional assessment in Xenopus laevis oocytes and expression in fish embryos.

Recently, we reported the cloning of a putative glucose transporter (OnmyGLUT1) from rainbow trout embryos. In this paper, we describe the functional characteristics of OnmyGLUT1 and its expression during embryonic development of rainbow trout. Transport of D-glucose was analysed in Xenopus laevis oocytes following microinjection of mRNA transcribed in vitro. These experiments confirmed that OnmyGLUT1 is a facilitative Na(+)-independent transporter. Assessment of substrate selectivity, sensitivity to cytochalasin B and phloretin and kinetic parameters showed that the rainbow trout glucose transporter was similar to a carp transporter and to mammalian GLUT1. Embryonic expression of OnmyGLUT1 was studied using whole-mount in situ hybridization. Ubiquitous distribution of transcripts was observed until the early phase of somitogenesis. During the course of organogenesis, somitic expression decreased along the rostro-caudal axis, finally ceasing in the mature somites. The OnmyGLUT1 transcripts were detected in the neural crest during the whole study period. Transcripts were also found in structures that are likely to originate from the neural crest cells (gill arches, pectoral fins, upper jaw, olfactory organs and primordia of mouth lips). Hexose transport activity was detected at all developmental stages after blastulation. Cytochalasin B blocked the accumulation of phosphorylated 2-deoxy-D-glucose by dissociated embryonic cells, suggesting an important role for transport in glucose metabolism.

Glucose sensor for low-cost lifetime-based sensing using a genetically engineered protein.

We describe a glucose sensor based on a mutant glucose/galactose binding protein (GGBP) and phase-modulation fluorometry. The GGBP from Escherichia coli was mutated to contain a single cysteine residue at position 26. When labeled with a sulfhydryl-reactive probe 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid, the labeled protein displayed a twofold decrease in intensity in response to glucose, with a dissociation constant near 1 microM glucose. The ANS-labeled protein displayed only a modest change in lifetime, precluding lifetime-based sensing of glucose. A modulation sensor was created by combining ANS26-GGBP with a long-lifetime ruthenium (Ru) metal-ligand complex on the surface of the cuvette. Binding of glucose changed the relative intensity of ANS26-GGBP and the Ru complex, resulting in a dramatic change in modulation at a low frequency of 2.1 MHz. Modulation measurements at 2.1 MHz were shown to accurately determine the glucose concentration. These results suggest an approach to glucose sensing with simple devices.

The use of the reverse transcription-competitive polymerase chain reaction to investigate the in vivo regulation of gene expression in small tissue samples.

Reverse transcription-polymerase chain reaction (RT-PCR) is widely used to detect low abundance mRNAs in small samples. Accurate quantitative measurement of their level, as required for the study of gene expression, can be performed by RT-competitive PCR, a method that relies on the addition of known amounts of a cDNA competitor molecule in the amplification reactions. Here we demonstrate that this method can be easily set up in any laboratory with a minimum of equipment in molecular biology, and that either homologous or heterologous competitor, with a small difference in sequence length relative to the target, can be used to quantify specific mRNA accurately. We propose the utilization of a thermostable reverse transcriptase in the RT step to overcome the problem of the efficiency of target cDNA synthesis. In addition, to obtain reliable measurements, we recommend performing four PCR reactions with amounts of competitor flanking the concentration of the target mRNA.

Assessment of glucose transporter gene expression using the polymerase chain reaction.

We used a novel adaptation of the polymerase chain reaction to examine relative levels of mRNA encoding two members of the facilitative glucose transporter gene family, the GLUT1 or erythrocyte/HepG2/brain isoform and the GLUT4 or insulin-regulatable isoform. The method was fast (vs. hybridization methods), required no specific probe, and used total RNA samples of less than 1 microgram. Taking advantage of regions of structural similarity and differences between the two isoforms, we designed a single set of oligonucleotide primers capable of amplifying both GLUT1 and GLUT4 cDNAs such that their respective products could be resolved on the basis of a 12 base pair size differential. Hence, reverse transcription and complementary DNA amplification could be carried out for both transcripts using identical primers in the same reaction tube. Using this methodology, we examined the relative amounts of GLUT4 and GLUT1 mRNAs in several rat tissues. As expected based on prior reports using Northern analysis, rat brain contained only GLUT1 mRNA and skeletal muscle contained a large predominance of GLUT4 mRNA. Both isoform mRNAs were found in adipose tissue whereas adipose cells, heart and diaphragm contained predominantly GLUT4 mRNA. Induction of diabetes with streptozocin decreased the GLUT4 to GLUT1 ratio in adipose tissue 4-fold and 24 h of insulin treatment of the diabetic rats increased this ratio 9- to 10-fold. Insulin treatment of normal rats increased this ratio by 70%. Hindlimb skeletal muscle GLUT4 mRNA was quantified in diabetic and insulin-treated diabetic rats as a function of brain GLUT1 mRNA added as an internal standard. Using this methodology, no significant difference in muscle GLUT4 mRNA was noted as a result of 24 h of insulin therapy. In summary, quantitative PCR may be used to compare mRNA levels encoding specific members of a gene family either within given cells or tissues or as affected by physiological perturbations. Subject to certain limitations discussed within, this methodology may be useful in future measurements of glucose transporter mRNA, especially when only small tissue or cell samples are available.