Glutamate efflux from rat brain slices and cultures: A comparison of the depolarizing agents potassium, 4-aminopyridine,and veratrine

The major excitatory amino acid neurotransmitter in the mammalian brain is glutamate (GLU). GLU release from nerve terminals is both calcium-dependent and-independent, yet these mechanisms of release are not fully understood. Potassium, 4-aminopyridine (4-AP) and veratrine are commonly used depolarizing agents that were studied for their ability to stimulate GLU efflux from brain slices. These agents produced significant regional variations in GLU efflux from rat brain slices. Potassium was the most potent of the three secretogogues tested. 4-AP produced a significant GLU efflux only in the cerebellum. Veratrine produced consistent stimulation of GLU efflux from all brain regions tested. Potassium was the only depolarizing agent tested that stimulated GLU release from primary astroglial cultures of rat cerebral cortex. All three agents also demonstrated an ability to inhibit GLU reuptake in brain slice preparations. This data suggest that both GLU release and uptake are modulated in a regionally selective manner, and that commonly used depolarizing agents affect not only calcium-dependent neuronal release, but also uptake and glial responses.     

Expression of bak in S. pombe results in a lethality mediated through interaction with the calnexin homologue Cnx1

Expression studies in the yeast S. pombe have been utilised to establish the basis for a genetic analysis designed to identify the lethal partners of the pro-apoptotic proteins bak and bax. Bak expression in S. pombe is lethal and this lethality is rescued by co-expression of bcl-2 or bcl-x(L). S. pombe cells expressing bak have a terminal phenotype in which the majority of cells are blocked in the G1 phase of the cell cycle while the remainder of cells, unable to complete M-phase, mis-coordinate the timing of subsequent events in the cell cycle. Although bax expression in S. pombe gives rise to a slow growth phenotype, not a lethality, bax expressing cells display the same cell cycle phenotypes described for bak. Electron microscopy of cells expressing bak reveals a dramatic accumulation of large vesicular structures. A two-hybrid screen designed to identify S. pombe proteins which interact with bak, isolated the S. pombe calnexin homologue cnx1. Genetic analysis demonstrates that the Cnx1 domain which binds to bak in two-hybrid experiments, is necessary for bak lethality in S. pombe. This report identifies a lethal interacting partner for bak and the observations suggest a model for bak mediated lethality which can be tested in higher cells.     

Multiplex PCR and Next Generation Sequencing for the Non-Invasive Detection of Bladder Cancer

Background

Highly sensitive and specific urine-based tests to detect either primary or recurrent bladder cancer have proved elusive to date. Our ever increasing knowledge of the genomic aberrations in bladder cancer should enable the development of such tests based on urinary DNA.

Methods

DNA was extracted from urine cell pellets and PCR used to amplify the regions of the TERT promoter and coding regions of FGFR3, PIK3CA, TP53, HRAS, KDM6A and RXRA which are frequently mutated in bladder cancer. The PCR products were barcoded, pooled and paired-end 2 x 250 bp sequencing performed on an Illumina MiSeq. Urinary DNA was analysed from 20 non-cancer controls, 120 primary bladder cancer patients (41 pTa, 40 pT1, 39 pT2+) and 91 bladder cancer patients post-TURBT (89 cancer-free).

Results

Despite the small quantities of DNA extracted from some urine cell pellets, 96% of the samples yielded mean read depths >500. Analysing only previously reported point mutations, TERT mutations were found in 55% of patients with bladder cancer (independent of stage), FGFR3 mutations in 30% of patients with bladder cancer, PIK3CA in 14% and TP53 mutations in 12% of patients with bladder cancer. Overall, these previously reported bladder cancer mutations were detected in 86 out of 122 bladder cancer patients (70% sensitivity) and in only 3 out of 109 patients with no detectable bladder cancer (97% specificity).

Conclusion

This simple, cost-effective approach could be used for the non-invasive surveillance of patients with non-muscle-invasive bladder cancers harbouring these mutations. The method has a low DNA input requirement and can detect low levels of mutant DNA in a large excess of normal DNA. These genes represent a minimal biomarker panel to which extra markers could be added to develop a highly sensitive diagnostic test for bladder cancer.     

Acute anticonvulsant activity of structural analogues of valproic acid and changes in brain GABA and aspartate content

Ten analogues of valproic acid (substituted butyric, pentanoic and hexanoic acids) were tested for anticonvulsant activity against audiogenic seizures in DBA/2 mice. There is a consistent correlation between the structure of these branched-chain fatty acids and their anticonvulsant potency, the larger molecules being the more active. There is also a strong correlation between the anticonvulsant potency of these compounds and their ability to reduce cerebral aspartate levels. Cerebral GABA levels are elevated by most, but not all, of the actively anticonvulsant valproate analogues.     

Changes in Regional Neurotransmitter Amino Acid Levels in Rat Brain During Seizures Induced by l-Allylglycine, Bicuculline, and Kainic Acid

Changes in amino acid concentrations were studied in the cortex, cerebellum, and hippocampus of the rat brain, after 20 min of seizure activity induced by kainic acid, 47 mumol/kg i.v.; L-allylglycine, 2.4 mmol/kg i.v.; or bicuculline, 3.27 mumol/kg i.v. in paralysed, mechanically ventilated animals. Metabolic changes associated with kainic acid seizures predominate in the hippocampus, where there are decreases in aspartate (-26%), glutamate (-45%), taurine (-20%), and glutamine (-32%) concentrations and an increase in gamma-aminobutyric acid (GABA) concentration (+ 26%). L-Allylglycine seizures are associated with generalized decreases in GABA concentrations (-32 to -54%), increases in glutamine concentrations (+10 to +53%), and a decrease in cortical aspartate concentration (-14%). Bicuculline seizures, in fasted rats, are associated with marked increases in the levels of hippocampal GABA (+106%) and taurine (+40%). In the cerebellum, there are increases in glutamine (+50%) and taurine concentrations (+36%). These changes can be explained partially in terms of known biochemical and neurophysiological mechanisms, but uncertainties remain, particularly concerning the cerebellar changes and the effects of kainic acid on dicarboxylic amino acid metabolism.     

GABA-like immunoreactivity of identified interneurons in the flight system of the locust,Locusta migratoria

Summary The transmitter content of identified inhibitory interneurons in the flight system of the locust, Locusta migratoria, has been characterized using antibodies raised against protein-conjugated gamma aminobutyric acid. Identified flight neurons were filled with the fluorescent dye, Lucifer Yellow. Serial sections of dye-filled neurons were incubated with an antibody to gamma aminobutyric acid which was subsequently tagged with a fluorescent marker. Excitatory motoneurons to wing muscles and 13 flight interneurons (3 excitatory, 7 inhibitory, and 3 with unknown synaptic effect) were examined. Neither the moto-neurons nor any of the 3 excitatory interneurons contained immunoreactive material. Six of the 7 inhibitory interneurons did contain immunoreactive material. All the neurons which contained immunoreactive material and whose synaptic effect is known were inhibitory. We conclude that most of the inhibitory flight interneurons which have been described use gamma aminobutyric acid as their transmitter. Interestingly, at least 1 set of interneurons known to be inhibitory does not use gamma aminobutyric acid. We predict that the 2 interneurons which do contain immunoreactive material and whose synaptic effect is not yet known will be found to have inhibitory roles in the operation of the flight circuitry.     

Premethylation of Foreign DNA Improves Integrative Transformation Efficiency in Synechocystis sp. Strain PCC 6803

Restriction digestion of foreign DNA is one of the key biological barriers against genetic transformation in microorganisms. To establish a high-efficiency transformation protocol in the model cyanobacterium, Synechocystis sp. strain PCC 6803 (Synechocystis 6803), we investigated the effects of premethylation of foreign DNA on the integrative transformation of this strain. In this study, two type II methyltransferase-encoding genes, i.e., sll0729 (gene M) and slr0214 (gene C), were cloned from the chromosome of Synechocystis 6803 and expressed in Escherichia coli harboring an integration plasmid. After premethylation treatment in E. coli, the integration plasmid was extracted and used for transformation of Synechocystis 6803. The results showed that although expression of methyltransferase M had little impact on the transformation of Synechocystis 6803, expression of methyltransferase C resulted in 11- to 161-fold-higher efficiency in the subsequent integrative transformation of Synechocystis 6803. Effective expression of methyltransferase C, which could be achieved by optimizing the 5′ untranslated region, was critical to efficient premethylation of the donor DNA and thus high transformation efficiency in Synechocystis 6803. Since premethylating foreign DNA prior to transforming Synechocystis avoids changing the host genetic background, the study thus provides an improved method for high-efficiency integrative transformation of Synechocystis 6803.