The intervention effect of licorice in d-galactose induced aging rats by regulating the taurine metabolic pathway.

Licorice, an edible and officinal plant material, has attracted considerable attention for its wide range of pharmacological activities. Our previous study showed that licorice can ameliorate cognitive damage and improve oxidative stress and apoptosis in aging rats induced by d-galactose (d-gal). In this study, in order to further explore the changes of the metabolic profile during the aging process and the antiaging mechanism of licorice, the 1H NMR-based metabolomics approach was used to analyze serum and urine samples and identify a potential biomarker in d-gal induced aging rats. The results revealed that the taurine metabolic pathway was significantly correlated with the ageing process in d-gal induced rats. Furthermore, the taurine contents were significantly decreased in both the serum and urine samples of aging rats compared with the controls. At the same time, the levels of cysteine dioxygenase type I (CDO1), cysteine sulfinic acid decarboxylase (CSAD) and glutamate decarboxylase type I (GAD1), which are the key enzymes affecting the synthesis reactions, were decreased in aging rats compared with the controls. After licorice administration, the levels of taurine, CDO1 and CSAD were all significantly increased. These findings firstly demonstrated that the regulation of the taurine metabolic pathway is involved in the anti-aging effect of licorice in d-gal induced aging rats.

Dual effects of slightly acidic electrolyzed water (SAEW) treatment on the accumulation of γ-aminobutyric acid (GABA) and rutin in germinated buckwheat.

In the present study, the dual effects of slightly acidic electrolyzed water (SAEW) treatment on γ-aminobutyric acid (GABA) and rutin accumulation of germinated buckwheat were evaluated during germination. The results showed that SAEW treatment (pH 5.83, ACC of 20.3 mg/L) could promote the accumulation of GABA and rutin in germinated buckwheat. The GABA and rutin contents of SAEW-germinated buckwheat reached 143.20 and 739.9 mg/100 g respectively, which is significantly higher than those of control (P<0.05). Moreover, SAEW treatment could increase the activity of glutamic acid decarboxylase (GAD) and phenylalanine ammonialyase (PAL) and thus result in the GABA and rutin accumulation of germinated buckwheat. The results suggested that SAEW treatment could promote the rutin accumulation of germinated buckwheat by influencing phenylpropanoid secondary metabolic pathway instead of the inhibition of rutin degrading enzyme (RDE) activity. In addition, SAEW treatment had no adverse impact on the sprouts growth and could reduce the microbial populations of germinated buckwheat during germination.

A downstream process allowing the efficient isolation of a recombinant amphiphilic protein from tobacco leaves.

The 65-kDa isoform of human glutamic acid decarboxylase (hGAD65) is a major autoantigen in autoimmune diabetes. The heterologous production of hGAD65 for diagnostic and therapeutic applications is hampered by low upstream productivity and the absence of a robust and efficient downstream process for product isolation. A tobacco-based platform has been developed for the production of an enzymatically-inactive form of the protein (hGAD65mut), but standard downstream processing strategies for plant-derived recombinant proteins cannot be used in this case because the product is amphiphilic. We therefore evaluated different extraction buffers and an aqueous micellar two-phase system (AMTPS) to optimize the isolation and purification of hGAD65mut from plants. We identified the extraction conditions offering the greatest selectivity for hGAD65mut over native tobacco proteins using a complex experimental design approach. Under our optimized conditions, the most efficient initial extraction and partial purification strategy achieved an overall hGAD65mut yield of 92.5% with a purification factor of 12.3 and a concentration factor of 23.8. The process also removed a significant quantity of phenols, which are major contaminants present in tobacco tissue. This is the first report describing the use of AMTPS for the partial purification of an amphiphilic recombinant protein from plant tissues and our findings could also provide a working model for the initial recovery and partial purification of hydrophobic recombinant proteins from transgenic tobacco plants.

Applications of small-angle X-ray scattering to biomacromolecular solutions.

Small-angle scattering of X-rays (SAXS) is an established method for low-resolution structural characterization of biological macromolecules in solution. Being complementary to the high resolution methods (X-ray crystallography and NMR), SAXS is often used in combination with them. The technique provides overall three-dimensional structures using ab initio reconstructions and hybrid modeling, and allows one to quantitatively characterize equilibrium mixtures as well as flexible systems. Recent progress in SAXS instrumentation, most notably, high brilliance synchrotron sources, has paved the way for high throughput automated SAXS studies allowing screening of external conditions (pH, temperature, ligand binding etc.). The modern approaches for SAXS data analysis are presented in this review including rapid characterization of macromolecular solutions in terms of low-resolution shapes, validation of high-resolution models in close-to-native conditions, quaternary structure analysis of complexes and quantitative description of the oligomeric composition in mixtures. Practical aspects of SAXS as a standalone tool and its combinations with other structural, biophysical or bioinformatics methods are reviewed. The capabilities of the technique are illustrated by a selection of recent applications for the studies of biological molecules. Future perspectives on SAXS and its potential impact to structural molecular biology are discussed.

Glutamate decarboxylase of the parasitic arthropods Ctenocephalides felis and Rhipicephalus microplus: gene identification, cloning, expression, assay development, identification of inhibitors by high throughput screening and comparison with the orthologs from Drosophila melanogaster and mouse.

Glutamate decarboxylase (l-glutamate 1-carboxylyase, E.C. 4.1.1.15, GAD) is the rate-limiting enzyme for the production of γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in vertebrates and invertebrates. We report the identification, isolation and characterization of cDNAs encoding GAD from the parasitic arthropods Ctenocephalides felis (cat flea) and Rhipicephalus microplus (cattle tick). Expression of the parasite GAD genes and the corresponding Drosophila melanogaster (fruit fly) GAD1 as well as the mouse GAD(65) and GAD(67) genes in Escherichia coli as maltose binding protein fusions resulted in functional enzymes in quantities compatible with the needs of high throughput inhibitor screening (HTS). A novel continuous coupled spectrophotometric assay for GAD activity based on the detection cascade GABA transaminase/succinic semialdehyde dehydrogenase was developed, adapted to HTS, and a corresponding screen was performed with cat flea, cattle tick and fruit fly GAD. Counter-screening of the selected 38 hit substances on mouse GAD(65) and GAD(67) resulted in the identification of non-specific compounds as well as inhibitors with preferences for arthropod GAD, insect GAD, tick GAD and the two mouse GAD forms. Half of the identified hits most likely belong to known classes of GAD inhibitors, but several substances have not been described previously as GAD inhibitors and may represent lead optimization entry points for the design of arthropod-specific parasiticidal compounds.

Expression, purification, and biochemical characterization of Mycobacterium tuberculosis aspartate decarboxylase, PanD.

Like all bacteria, Mycobacterium tuberculosis (Mtb) possesses the genes necessary for coenzyme A biosynthesis and metabolism. In the present work, the Mtb panD gene was PCR amplified, overexpressed, and purified by metal affinity chromatography. The recombinant Mtb panD was found to exist as a tetramer in solution. Incubation of Mtb panD at 37 degrees C for several hours resulted in a complete cleavage of the inactive (pi) form into the two subunits (alpha and beta). The cleavage was confirmed by Western blot analysis as well as by N-terminal sequencing. Cleaved Mtb panD was assayed for decarboxylase activity with L-aspartate as substrate. The kinetic parameters K(m) and k(cat) were found to be 219 microM and 0.65s(-1), respectively. These results provide the means for further studies based on the identification of the Mtb panD as well as other components of pantothenate metabolism as potential drug targets.

Cloning and nucleotide sequence of the glutamate decarboxylase-encoding gene gadA from Aspergillus oryzae.

We cloned a genomic DNA encoding the glutamate decarboxylase (GAD) from Aspergillus oryzae using a 200-bp DNA fragment as the probe. This DNA fragment was amplified by the reverse transcription polymerase chain reaction with mRNA of A. oryzae as the template and degenerate primers designed from the conserved amino acid sequence of Escherichia coli GAD and Arabidopsis thaliana GAD. Nucleotide sequencing analysis showed that the cloned gene (designated gadA) encoded 514 amino acid residues and contained three introns. Southern hybridization showed that the gadA gene was on a 6.0-kb SacI fragment and that there was a single copy in the A. oryzae chromosome. The cloned gene was functional, because one transformant of A. oryzae containing multiple copies of the gadA gene had 10-fold the GAD activity and a 12-fold increase in gamma-aminobutyric acid production compared with the control strain.

Subthalamic GAD gene transfer in Parkinson disease patients who are candidates for deep brain stimulation.

This gene transfer experiment is the first Parkinson's Disease (PD) protocol to be submitted to the Recombinant DNA Advisory Committee. The principal investigators have uniquely focused their careers on both pre-clinical work on gene transfer in the brain and clinical expertise in management and surgical treatment of patients with PD. They have extensively used rodent models of PD for proof-of-principle experiments on the utility of different vector systems. PD is an excellent target for gene therapy, because it is a complex acquired disease of unknown etiology (apart from some rare familial cases) yet it is characterized by a specific neuroanatomical pathology, the degeneration of dopamine neurons of the substantia nigra (SN) with loss of dopamine input to the striatum. This pathology results in focal changes in the function of several deep brain nuclei, which have been well-characterized in humans and animal models and which account for many of the motor symptoms of PD. Our original approaches, largely to validate in vivo gene transfer in the brain, were designed to facilitate dopamine transmission in the striatum using an AAV vector expressing dopamine-synthetic enzymes. Although these confirmed the safety and potential efficacy of AAV, complex patient responses to dopamine augmenting medication as well as poor results and complications of human transplant studies suggested that this would be a difficult and potentially dangerous clinical strategy using current approaches. Subsequently, we and others investigated the use of growth factors, including GDNF. These showed some encouraging effects on dopamine neuron survival and regeneration in both rodent and primate models; however, uncertain consequences of long-term growth factor expression and question regarding timing of therapy in the disease course must be resolved before any clinical study can be contemplated. We now propose to infuse into the subthalamic nucleus (STN) recombinant AAV vectors expressing the two isoforms of the enzyme glutamic acid decarboxylase (GAD-65 and GAD-67), which synthesizes the major inhibitory neurotransmitter in the brain, GABA. The STN is a very small nucleus (140 cubic mm or 0.02% of the total brain volume, consisting of approximately 300,000 neurons) which is disinhibited in PD, leading to pathological excitation of its targets, the internal segment of the globus pallidus (GPi) and substantia nigra pars reticulata (SNpr). Increased GPi/SNpr outflow is believed responsible for many of the cardinal symptoms of PD, i.e., tremor, rigidity, bradykinesia, and gait disturbance. A large amount of data based on lesioning, electrical stimulation, and local drug infusion studies with GABA-agonists in human PD patients have reinforced this circuit model of PD and the central role of the STN. Moreover, the closest conventional surgical intervention to our proposal, deep brain stimulation (DBS) of the STN, has shown remarkable efficacy in even late stage PD, unlike the early failures associated with recombinant GDNF infusion or cell transplantation approaches in PD. We believe that our gene transfer strategy will not only palliate symptoms by inhibiting STN activity, as with DBS, but we also have evidence that the vector converts excitatory STN projections to inhibitory projections. This additional dampening of outflow GPi/SNpr outflow may provide an additional advantage over DBS. Moreover, of perhaps the greatest interest, our preclinical data suggests that this strategy may also be neuroprotective, so this therapy may slow the degeneration of dopaminergic neurons. We will use both GAD isoforms since both are typically expressed in inhibitory neurons in the brain, and our data suggest that the combination of both isoforms is likely to be most beneficial. Our preclinical data includes three model systems: (1) old, chronically lesioned parkinsonian rats in which intraSTN GAD gene transfer results not only in improvement in both drug-induced asymmetrical behavior (apomorphine symmetrical rotations), but also in spontaneous behaviors. In our second model, GAD gene transfer precedes the generation of a dopamine lesion. Here GAD gene transfer showed remarkable neuroprotection. Finally, we carried out a study where GAD-65 and GAD-67 were used separately in monkeys that were resistant to MPTP lesioning and hence showed minimal symptomatology. Nevertheless GAD gene transfer showed no adverse effects and small improvements in both Parkinson rating scales and activity measures were obtained. In the proposed clinical trial, all patients will have met criteria for and will have given consent for STN DBS elective surgery. Twenty patients will all receive DBS electrodes, but in addition they will be randomized into two groups, to receive either a solution containing rAAV-GAD, or a solution which consists just of the vector vehicle, physiological saline. Patients, care providers, and physicians will be blind as to which solution any one patient receives. All patients, regardless of group, will agree to not have the DBS activated until the completion and unblinding of the study. Patients will be assessed with a core clinical assessment program modeled on the CAPSIT, and in addition will also undergo a preop and several postop PET scans. At the conclusion of the study, if any patient with sufficient symptomatic improvement will be offered DBS removal if they so desire. Any patients with no benefit will simply have their stimulators activated, which would normally be appropriate therapy for them and which requires no additional operations. If any unforeseen symptoms occur from STN production of GABA, this might be controlled by blocking STN GABA release with DBS, or STN lesioning could be performed using the DBS electrode. Again, this treatment would not subject the patient to additional invasive brain surgery. The trial described here reflects an evolution in our thinking about the best strategy to make a positive impact in Parkinson Disease by minimizing risk and maximizing potential benefit. To our knowledge, this proposal represents the first truly blinded, completely controlled gene or cell therapy study in the brain, which still provides the patient with the same surgical procedure which they would normally receive and should not subject the patient to additional surgical procedures regardless of the success or failure of the study. This study first and foremost aims to maximally serve the safety interests of the individual patient while simultaneously serving the public interest in rigorously determining in a scientific fashion if gene therapy can be effective to any degree in treating Parkinson's disease.

Rice (Oryza sativa) contains a novel isoform of glutamate decarboxylase that lacks an authentic calmodulin-binding domain at the C-terminus.

We have isolated full-length cDNAs for two distinct isoforms of glutamate decarboxylase (GAD), designated OsGAD1 and OsGAD2 from a rice shoot cDNA library. Open reading frames found in OsGAD1 and OsGAD2 cDNAs encode putative proteins of 501 (56.7 kDa) and 500 amino acids (55.6 kDa), respectively. They show 69% identity to each other and 67-78% identity to dicotyledonous counterpart sequences determined so far. Comparative analysis of relevant genomic clones obtained from the rice genomic library with these cDNAs as probes demonstrated that the number and sizes of introns deduced for these two genes differ considerably. Interestingly, in the regions in the putative gene products corresponding to the C-terminal 30-amino-acid peptide known as the calmodulin-binding domain of plant GADs, OsGAD1 possesses a typical motif, while OsGAD2 contains several substitutions of amino acids that contribute strongly to the binding of calmodulin (CaM). An in vitro CaM-binding assay of these proteins over-expressed in Escherichia coli revealed that OsGAD1 can in fact bind specifically to bovine CaM but OsGAD2 cannot. RNA analysis showed that transcripts of OsGAD1 and OsGAD2 were present in all tissues examined, but their expression was differentially regulated, at least in roots and maturing seeds.

Expression of the Corynebacterium glutamicum panD gene encoding L-aspartate-alpha-decarboxylase leads to pantothenate overproduction in Escherichia coli.

The Corynebacterium glutamicum panD gene was identified by functional complementation of an Escherichia coli panD mutant strain. Sequence analysis revealed that the coding region of panD comprises 411 bp and specifies a protein of 136 amino acid residues with a deduced molecular mass of 14.1 kDa. A defined C. glutamicum panD mutant completely lacked L-aspartate-alpha-decarboxylase activity and exhibited beta-alanine auxotrophy. The C. glutamicum panD (panDC. g.) as well as the E. coli panD (panDE.c.) genes were cloned into a bifunctional expression plasmid to allow gene analysis in C. glutamicum as well as in E. coli. The enhanced expression of panDC.g. in C. glutamicum resulted in the formation of two distinct proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, leading to the assumption that the panDC.g. gene product is proteolytically processed into two subunits. By increased expression of panDC.g. in C. glutamicum, the activity of L-aspartate-alpha-decarboxylase was 288-fold increased, whereas the panDE.c. gene resulted only in a 4-fold enhancement. The similar experiment performed in E. coli revealed that panDC.g. achieved a 41-fold increase and that panDE.c. achieved a 3-fold increase of enzyme activity. The effect of the panDC.g. and panDE.c. gene expression in E. coli was studied with a view to pantothenate accumulation. Only by expression of the panDC.g. gene was sufficient beta-alanine produced to abolish its limiting effect on pantothenate production. In cultures expressing the panDE.c. gene, the maximal pantothenate production was still dependent on external beta-alanine supplementation. The enhanced expression of panDC.g. in E. coli yielded the highest amount of pantothenate in the culture medium, with a specific productivity of 140 ng of pantothenate mg (dry weight)-1 h-1.