GABAA receptors as novel drug targets for treatment of mental disorders

A balance between excitatory and inhibitory neurotransmissions in brain is an essential factor for the proper function of the brain. The amino acid gamma-aminobutyric-acid (GABA) is considered as the major inhibitory neurotransmitter in brain. Thus, GABAergic neurons play a key role in regulating behavior. Previous data have revealed the complex subunit structural design for GABAA receptor channel, in which a pentameric assembly resulting from 5 of at least 21 subunits, grouped in the eight classes alpha (α1-6), beta (β1-4), gamma (γ1-4), delta, pi (π), epsilon (ε), theta (θ) and rho (ρ1-3) permits an immense number of putative receptor isoforms. GABAARs are highly diversed in the central nervous system in which this diversity may be related to some mental disorders. Any alteration in expression of the GABAA receptor genes causes neurophysiological and functional consequences that might be associated with neurological disorders. Some neuropsychiatric disorders, such as anxiety, epilepsy and sleep disorders, are effectively treated with therapeutic agents that act on the GABAA receptor. In this article, the contribution of GABAA receptor deficits to central nervous system disorders, in particular anxiety disorders, epilepsy, schizophrenia and insomnia, will be reviewed. The better understanding of GABA and its receptors may help us to find novel therapeutic agents for treatment of mental disorder in future research

Molecular gene cloning and sequencing of glutamate decarboxylase gene from Lactobacillus delbrueckii and Lactobacillus reuteri

     Glutamate decarboxylase enzyme produces γ-aminobutyric acid (GABA) in a non-reversible decarboxylation reaction of glutamate. GABA is a major inhibitory neurotransmitter of the brain and it is also present at high concentration in other organs such as pancreatic islets. GABA has effects on blood pressure, diabetes, inflammation, sleeplessness and depression. Some bacteria such as Lactobacillus strains are capable of GABA production. Identification of these bacteria is important both for researchers and industry. The aim of this study was molecular gene cloning and sequencing of glutamate decarboxylase (gad) from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 and Lactobacillus reuteri ATCC 23272. These strains were cultured in MRS medium at 37○C for 24 hours. For cloning gad gene from these strains, polymerase chain reaction (PCR) was performed using specific primers designed by Oligo7 software. PCR production was extracted from agarose gel and was inserted into PGEM-T vector using T4 DNA ligase enzyme and then it was transformed to E. coli XL1Blue. In the final step, white colonies were selected and after plasmid extraction, the existence of gad gene in recombinants was confirmed by PCR. Gad gene was cloned from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 and Lactobacillus reuteri ATCC 23272. It is for the first time that the gad gene sequences from these bacteria were registered on NCBI with accession numbers KF751355 and KF751352 respectively. The result of this research indicates that the two aforementioned bacteria contain glutamate decarboxylase gene and therefore they possibly can be used for industrial γ-aminobutyric acid production

Bioinformatics designing of 10-23 deoxyribozyme against noncoding region before start codon of beta-galactosidase gene (lacZ) in pGEM-T vector

Background and aims: Deoxyribozymes are oligoribodeoxynucleotides that catalyze reactions such as cutting RNA and have diagnostic and therapeutic applications. Deoxyribozyme 10-23 includes a catalytic domain dependent on a fixed 15-nucleotic (mer) cation and two variable binding arms that cause the specificity of enzymes. Lactose operon is used in the white-blue screening process. This operon includes three polycistronic genes. In this study, a deoxyribozyme against α-peptide beta-galactosidase gene in the lactose operon was designed. Methods: pGEM-T map was obtained from addgene server and α-peptide gene sequence was determined. Then, using expasy website proper protein frame in comparison with various reading frames was determined. In this step, whole sequence was reversed and mRNA sequence was achieved. Secondary structure with the lowest free energy was gained using mfold server. Considering the fact that 10-23 deoxyribozyme has cutting capability between a unpaired purine and pairs pyrimidine; an AC was selected in ribosome binding site in the untranslated region and then 9 open bases on either side of it was used as a binding arms. Investigation of the absence of similar sequences in host bacteria was performed by NCBI server. Finally, activity and binding of deoxyribozyme was predicted by the mfold server. Results: The results of this study showed that the designed deoxyribozyme had a relatively high Tm with two 9-nucleotide arms, which increased its effectiveness. Conclusion: The results of this study can be used to control the expression of lacZ gene as a biomarker

Salt overly sensitive 1 (SOS1) gene expression can be regulated via Azospirillum brasilense Sp7 in wheat seedlings under saline condition

Salinity stress reduces plant growth via failure of physiological processes mainly due to the abundance of Na+ ion. Salt overly sensitive (SOS) signaling pathway is considered as an important component of Na+/K+ homeostasis system in plants, especially under saline condition. Moreover, it is reported that wheat-Azospirillum associated has resulted in an enhanced salinity tolerance. To evaluate involvement of Azospirillum species in regulation of SOS signaling pathway, inoculated and none-inoculated wheat seedlings with Azospirillum brasilense Sp7 were grown for five days. Then uniform seedlings were transferred into saline hydroponic media with and without 200 mM NaCl. The relative expression of TaSOS1 of root, sheath, and blade as well as Na+/K+ ratio was measured after 6, 24 and 48 hours since inoculated and non-inoculated seedling were transferred to NaCl media. Simultaneously Ca, Fe, proline content, root and shoot dry mass and soluble sugars were measured at 72 hour after application of NaCl. Result showed that salinity increased TaSOS1 gene expression, Na+, prolin and Na+/K+ ratio but Ca and Fe were decreased in root and shoot of wheat seedlings. Although A. brasilense Sp7 could improve salinity tolerance in wheat via reduction of Na uptake and upregulation of TaSOS1 expression, but do not have any effect in sodium distribution within plant parts. Therefore, salinity could increase TaSOS1 expression in the root, sheath and blade and A. brasilense Sp7 also could reduce the adverse effect of salinity via addition of over expression of TaSOS1.</p

Application of dietary copepod, Acanthocyclops trajani in the first feeding of beluga larvae (Huso huso)

This study was carried out regarding the effects of cyclopoid copepod Acanthocyclops trajani on first feeding of beluga larvae (Huso huso). The treatments comprised a control diet containing Artemia naupli and Daphnia magna and a combined diet containing Artemia naupli, Daphnia magna and A. trajani. The results of this study indicated that the average length, body weight and specific growth rate in larvae of control and combined diets were not significantly different. In spite of the fact that, A. trajani was significantly different in the amount of n-3 fatty acids in comparison with the other live feeds in this study, but indicated more effectiveness in survival rates (80%) of beluga larvae, as a supplement. Furthermore, the combined diet was significantly different in DHA, in comparison with control diet. This study indicated that freshwater copepod is potential supplemental live food, to increase nutritional value from the point of view in terms of essential fatty acids and survival rate of valuable fish larvae such as beluga

Designing a light-activated recombinant alpha hemolysin for colorectal cancer targeting

Introduction: Colorectal cancer (CRC) is one of the main health burden worldwide, which can cause major economic and physiological problems along with relatively high rate of mortality. It is important to develop new methods for the localized delivery of recombinant protein therapeutics, in large part due to the failure of conventional therapies in most cases. Since E. coli Nissle 1917 (EcN) does not produce any virulence factors, here we used these bacteria with the light-activated promoter system to deliver therapeutic agents in the desired location and time. Methods: In this study, Staphylococcus aureus alpha hemolysin (SAH), after codon usage optimization, was cloned into blue light activating vector (pDawn) and transferred to EcN strain. Then, the functionality and cytotoxicity of secreted alpha hemolysin was evaluated in the SW480 colon cancer cell line by using different experiments, including blood agar test, flow cytometry analysis, and DAPI staining. Results: Our findings revealed that EcN can produce functional SAH under the blue light irradiation against SW480 cancer cells. Moreover, cytotoxicity assays confirmed the dose- and time-dependent toxicity of this payload (SAH) against SW480 cancer cells. Conclusion: Based on our results, EcN is proposed as an appropriate light-activated vehicle for delivery of anticancer agents to the target cancer cells/tissues

Comparative effects of the arachidonic acid enrichment of the concentrated food and live feed on growth rate and reproductive indices in zebrafish, Danio rerio

In this study, the comparative effects of arachidonic acid (ARA) enrichment of the concentrated food and live feed (Artemia nauplius) were investigated on growth rate and reproductive indices in zebrafish (Danio rerio). Thirty-day-old juveniles (n = 400, with an average initial weight of 0.125 g) were randomly distributed in six treatments (each in three replicates) in eighteen 10-L aquariums (with a stocking density of 15 juveniles per aquarium). Fish were fed with concentrated diet containing three levels of ARA (0, 1 and 2% in the diet) and live feed (Instar II Artemia nauplii) in three levels of ARA (0, enriched with 1% and 2% of ARA) for 60 days until sexual maturity. The results of Two-Way ANOVA showed that the final body weight, body weight gain rate (%), and female’s reproductive indices were not affected by the combined effects of two factors; food type and ARA levels (p>0.05). Body weight in fish fed with concentrated diet containing 2% ARA was significantly higher than compared with the other treatments (p<0.05). The highest absolute and relative fecundities, and gonadosomatic index were obtained in females fed ARA-free concentrated diet, the enriched- Artemia with 1-2% ARA, and the enriched- Artemia with 1% ARA (p<0.05), respectively. The ARA enrichment in the concentrated diet only accelerated the growth rate of fish until sexual maturity for breeding. However, fish fed the enriched- Artemia with 1% ARA exhibited higher gonadal weight and much higher relative fecundity compared to other groups despite lower final weight (p<0.05). Based on the results and in order to improve the female’s reproductive performance in zebrafish hatcheries, it is suggested that fish fed the enriched Artemia with 1% ARA two months before reproduction

DISTRIBUTION AND FUNCTION OF GABAA RECEPTOR SUBUNITS IN THE AMYGDALA

The amygdala has an important role in processing emotional information. Inappropriate processing within the amygdala is thought to induce anxiety disorders. Benzodiazepines, which act specifically on GABAA receptors, are commonly used as anxiolytics. This implies that GABAergic synapses within the amygdala may have an important role in inducing or compensating for these disorders. This thesis investigates the distribution and function of the GABAA receptor subunits in the amygdala. This study has made use of the real-time PCR, western blotting, and molecular cloning methods to determine expression levels of GABAA receptor subunits mRNAs and proteins and making recombinant GABAA receptors. We also used whole cell patch pipette recording to record the currents from transfected HEK 293 cells and acute brain slices containing amygdala. We examined the benzodiazepine pharmacology and subunit composition of the GABAA receptor subunits in the lateral and central amygdala. We examined the pharmacology of GABAA receptors by expressing different subunit combinations in HEK 293 cells and comparing the pharmacology with specific GABAergic inputs in the amygdala. Realtime PCR results showed that the GABAA receptor subunits are distributed differentially within the amygdalar nuclei with &#945;2, &#945;4, &#946;2, &#946;3, &#947;1 and &#947;2 abundant. In the case of the &#947; subunits, the expression level of the &#947;1 subunit was higher in the CeA whereas the expression level of &#947;2 was at high level in the LA. The &#945;1 subunit was at very low level in the CeA and &#945;2 was expressed at high level in the LA than in the CeA. The differences in the expression of &#945;1, &#945;2, &#947;1 and &#947;2 subunits in the amygdala suggest a diversity function for these subunits. Whole cell recording of HEK 293 cells showed that diazepam, zolpidem had different effects in these receptors depending on their compositions. Diazepam and zolpidem had low affinity for &#947;1-containing receptors. DMCM blocked the actions of GABA at &#945;1&#946;1&#947;2 and &#945;2&#946;1&#947;2 combinations (75 % current reduction) but had no effect at &#945;1&#946;1&#947;1 or &#945;2&#946;1&#947;1. In slice recordings DMCM blocked IPSCs by 70% in the lateral amygdala. In the central amygdala, DMCM had variable effects. Diazepam enhanced the amplitude of IPSC in the LA whereas the response in the CeA was either reduction or enhancement of currents. Similar results were obtained with zolpidem. Real-time PCR and whole cell recording from acute brain slices revealed a difference in the distribution of GABAA receptor subunits between the lateral and central amygdala. Our investigation showed there are sex differences in localisation of the GABAA receptor subunits in the rat amygdala in which the expression level of the &#945;2 and &#947;2 subunits might be higher in female rats, whereas the expression level of &#947;1 was higher in males than in females. We conclude that in the lateral amygdala all inputs have &#947;2 subunits whereas in the central amygdala some inputs contain &#947;1 subunit while others contain &#947;2 subunits

The Role of Atypical Chemokine Receptor CCXCKR (CCRL1) in Human Diseases

The role of chemokines and their receptors have been identified in many biological activities such as immune response and angiogenesis; however, their regulatory ways are under investigation. In recent years, homologous proteins’ typical chemokine receptors have been identified. Despite the structural similarity due to changes in a particular motif, they are not able to create signals through G proteins within the cell; therefore, these chemokine receptors are called silent or unusual. Through binding and internalization and degradation of the chemokines, these receptors regulate the level of ligand in the environment. In this paper, about fifty articles published in the field of the regulatory role of CCX-CKR receptor in some human diseases such as cancer and inflammatory diseases were reviewed. CCX-CKR is one of the chemokine binding proteins that like other silent chemokine receptors, is not able to induce intracellular signaling pathways. Since chemokine network plays a major role in many diseases such as cancer and autoimmune diseases through the impact of CCX-CKR on the level of chemokine environment, reventing intracellular signal creation by these proteins can be considered as a therapeutic target

Partial Cloning and Nucleotide Sequencing of Glutamate Decarboxylase Gene Isoform 65 from Human Brain

Background: Gamma -aminobutyric acid (GABA), a non-protein amino acid acts as an inhibitory neurotransmitter in the central nervous system of mammalians. The glutamate decarboxylase (GAD) is responsible for the conversion of L-glutamate to GABA. The human brain has two isoforms of this enzyme, GAD65 and GAD67 that differ in molecular weight, amino acid sequence, antigenicity, cellular location and interaction by factor of pyridoxal phosphate. The purpose of this study was cloning of gene encoding the human glutamate decarboxylase. Materials and Methods: Total cellular RNA was extracted from human brain tissue and then converted to cDNA. PCR was performed using exclusive primers for gad gene amplification. After purification of PCR product, it was partially cloned successfully in pJET1.2 blunt t-vector and was sent for sequencing. Results: The outcomes indicate that only gad gene was cloned partially. The length of human gad gene isoform 65 is 1759 base pair that encodes 585 amino acids. The length of partially cloned gad gene in this study was 385 base pair. Conclusion: Because obtaining fresh human brain is difficult and amount of mRNA is low, it may not be easy to clone full length of human gad gene. The approach described in this paper may be useful in cloning of other genes for which the corresponding mRNA is present at low levels