Tyrosine Hydroxylation in Betalain Pigment Biosynthesis Is Performed by Cytochrome P450 Enzymes in Beets (Beta vulgaris)

Yellow and red-violet betalain plant pigments are restricted to several families in the order Caryophyllales, where betacyanins play analogous biological roles to anthocyanins. The initial step in betalain biosynthesis is the hydroxylation of tyrosine to form L-DOPA. Using gene expression experiments in beets, yeast, and Arabidopsis, along with HPLC/MS analysis, the present study shows that two novel cytochrome P450 (CYP450) enzymes, CYP76AD6 and CYP76AD5, and the previously described CYP76AD1 can perform this initial step. Co-expressing these CYP450s with DOPA 4,5-dioxygenase in yeast, and overexpression of these CYP450s in yellow beets show that CYP76AD1 efficiently uses L-DOPA leading to red betacyanins while CYP76AD6 and CYP76AD5 lack this activity. Furthermore, CYP76AD1 can complement yellow beetroots to red while CYP76AD6 and CYP76AD5 cannot. Therefore CYP76AD1 uniquely performs the beet R locus function and beets appear to be genetically redundant for tyrosine hydroxylation. These new functional data and ancestral character state reconstructions indicate that tyrosine hydroxylation alone was the most likely ancestral function of the CYP76AD alpha and beta groups and the ability to convert L-DOPA to cyclo-DOPA evolved later in the alpha group.     

Pressure excursions during oscillatory flow in a branching network of tubes

The pressure difference across individual branches of a four-generation network of branching tubes was measured with the objective of obtaining general laws to describe the pressure drop in the airways under conditions of oscillatory flow. Fourier decomposition showed that the pressure signals consisted of a dominant component at the excitation frequency ("fundamental") and a "first harmonic" of smaller magnitude. For values of the ratio Re/alpha less than 200, the fundamental mainly represented fluid acceleration, whereas the first harmonic reflected the effects both of viscous dissipation and the change in total cross-sectional area between parent and daughter generations. For values of Re/alpha greater than 200, the magnitude of the fundamental was considerably larger than that due to fluid acceleration alone, suggesting the possibility of onset of turbulence in the branching network. These pressure measurements were applied to a simple model of the dog lung to predict total airway resistance. The results are found to be in substantial agreement with physiological measurements.     

Nonradioactive DNA detection on Southern blots by enzymatically triggered chemiluminescence

A chemiluminescent reaction based on the deprotection of a phosphorylated phenyl dioxetane by alkaline phosphatase has recently been described (Schaap, A.P., 1988, J. Biolumin. Chemilumin. 2, 253). Light output is enhanced by intermolecular energy transfer to a micelle-solubilized fluorophore. This system is applied here to the detection of DNA probes on Southern blots. Enzyme solution assays which give an indication of sensitivity show that using this substrate 100 fg (0.7 amol) alkaline phosphatase can be detected on a luminescence plate reader (200 ms reading time). In a model Southern blotting system 180 fg HindIII digested lambda DNA was detected on film with homologous biotinylated DNA and a streptavidin-alkaline phosphatase complex. The single copy genes mos and raf-1, representing targets of 4.2 and 2.4 pg target DNA respectively, have also been detected in Southern-blotted human genomic DNA. A delay in reaching a plateau level of light output which is dependent on pH is observed but signal continues for at least 7 days. Typically, 12-h exposures to X-ray film were performed but once a steady-state light output had been achieved this time could be reduced to 2 h by preflashing film. This detection system represents a sensitive nonradioactive method, which is applicable not only to Southern blots but also to Northern and Western blots and any assay in which alkaline phosphatase is the label.     

Implementation of reflex loops in a biomechanical finite element model

In the field of biomechanics, the offer of models which are more and more realistic requires to integrate a physiological response, in particular, the controlled muscle bracing and the reflexes. The following work aims to suggest a unique methodology which couples together a sensory and motor loop with a finite element model. Our method is applied to the study of the oscillation of the elbow in the case of a biceps brachial stretch reflex. The results obtained are promising in the purpose of the development of reactive human body models.     

Homology Modeling and Molecular Docking Studies of Glutamate Dehydrogenase (GDH) from Cyanobacterium Synechocystis sp. PCC 6803

Glutamate dehydrogenase (GDH), which is present in most bacteria and eukaryotes’ mitochondria, plays an important role in amino acid metabolism. In g     

The Effect of Carrier Distribution on Performance of ENZ-Based Electro-Absorption Modulator

Recently, epsilon-near-zero (ENZ) has been emerging as an important field of research which is the study of light-matter interactions in the presence of materials with zero permittivity. Since in many scientific works the uniform model of carrier distribution of Indium tin oxide (ITO) has been utilized, we want to investigate ENZ effect in ITO material and the effect of accurate carrier distribution on the performance of a modulator. For this reason, an electro-absorption (EA) modulators with a new configuration based on silicon slot modulator with indium thin oxide material is proposed. To study the effect of ENZ effect in ITO, the semiconductor model (realistic model) is utilized to model the carrier distribution in the ITO material. In this model, there is not any assumption. As a result, by applying the gate voltage, the insertion loss is increased 1.61 dB/μm in comparison with unbiased conditions. Also, the uniform model is used. Compared with the realistic model, the extinction ratio and figure-of-merit significantly enhance based on the uniform model, but the trends of results like insertion loss are so far from the realistic model. It can be found that the realistic model is reliable and the results are closer to reality.

     

Cell engineering method using fluorogenic oligonucleotide signaling probes and flow cytometry

Objective

Chromovert® Technology is presented as a new cell engineering technology to detect and purify living cells based on gene expression.

Methods

The technology utilizes fluorogenic oligonucleotide signaling probes and flow cytometry to detect and isolate individual living cells expressing one or more transfected or endogenously-expressed genes.

Results

Results for production of cell lines expressing a diversity of ion channel and membrane proteins are presented, including heteromultimeric epithelial sodium channel (αβγ-ENaC), sodium voltage-gated ion channel 1.7 (NaV1.7-αβ1β2), four unique γ-aminobutyric acid A (GABA_A) receptor ion channel subunit combinations α1β3γ2s, α2β3γ2s, α3β3γ2s and α5β3γ2s, cystic fibrosis conductance regulator (CFTR), CFTR-Δ508 and two G-protein coupled receptors (GPCRs) without reliance on leader sequences and/or chaperones. In addition, three novel plasmid-encoded sequences used to introduce 3′ untranslated RNA sequence tags in mRNA expression products and differentially-detectable fluorogenic probes directed to each are described. The tags and corresponding fluorogenic signaling probes streamline the process by enabling the multiplexed detection and isolation of cells expressing one or more genes without the need for gene-specific probes.

Conclusions

Chromovert technology is provided as a research tool for use to enrich and isolate cells engineered to express one or more desired genes.

     

Gap junction blockers: a potential approach to attenuate morphine withdrawal symptoms

Background

The exact mechanisms of morphine-induced dependence and withdrawal symptoms remain unclear. In order to identify an agent that can prevent withdrawal syndrome, many studies have been performed. This study was aimed to evaluate the effect of gap junction blockers; carbenoxolone (CBX) or mefloquine (MFQ); on morphine withdrawal symptoms in male rat.Adult male Wistar rats (225 – 275 g) were selected randomly and divided into 10 groups. All groups underwent stereotaxic surgery and in order to induce dependency, morphine was administered subcutaneously) Sc) at an interval of 12 hours for nine continuous days. On the ninth day of the experiment, animals received vehicle or CBX (100, 400, 600 μg/10 μl/rat, icv) or MFQ (50, 100 and 200 μg/10 μl/rat, icv) after the last saline or morphine (Sc) injection. Morphine withdrawal symptoms were precipitated by naloxone hydrochloride 10 min after the treatments. The withdrawal signs including: jumping, rearing, genital grooming, abdomen writhing, wet dog shake and stool weight, were recorded for 60 minutes.

Results

Results showed that CBX and MFQ decreased all withdrawal signs; and the analysis indicated that they could attenuate the total withdrawal scores significantly.

Conclusion

Taking together it is concluded that gap junction blockers prevented naloxone-precipitated withdrawal symptoms.