Expression of AMPA receptor subunits at synapses in laminae I–III of the rodent spinal dorsal horn

Neuropathic pain may arise following peripheral nerve injury though the molecular mechanisms associated with this are unclear. We used proteomic profiling to examine changes in protein expression associated with the formation of hyper-excitable neuromas derived from rodent saphenous nerves. A two-dimensional difference gel electrophoresis (2D-DIGE) profiling strategy was employed to examine protein expression changes between developing neuromas and normal nerves in whole tissue lysates. We found around 200 proteins which displayed a >1.75-fold change in expression between neuroma and normal nerve and identified 55 of these proteins using mass spectrometry. We also used immunoblotting to examine the expression of low-abundance ion channels Nav1.3, Nav1.8 and calcium channel α2δ-1 subunit in this model, since they have previously been implicated in neuronal hyperexcitability associated with neuropathic pain. Finally, S³⁵methionine in vitro labelling of neuroma and control samples was used to demonstrate local protein synthesis of neuron-specific genes. A number of cytoskeletal proteins, enzymes and proteins associated with oxidative stress were up-regulated in neuromas, whilst overall levels of voltage-gated ion channel proteins were unaffected. We conclude that altered mRNA levels reported in the somata of damaged DRG neurons do not necessarily reflect levels of altered proteins in hyper-excitable damaged nerve endings. An altered repertoire of protein expression, local protein synthesis and topological re-arrangements of ion channels may all play important roles in neuroma hyper-excitability.     

Phytochrome control of phototropism and chlorophyll accumulation in the apical cells of protonemal filaments of wildtype and an aphototropic mutant of the moss Ceratodon purpureus

The aphototropic mutant line ptr116 of the moss Ceratodon purpureus shows characteristics of a deficiency in the phytochrome chromophore. Photoreversibility measurements indicate an approximately 20 time lower concentration of spectrally active phytochrome compared to wild-type, whereas normal phytochrome apoprotein levels are found on immunoblots. Feeding with the tetrapyrroles biliverdin, the proposed precursor of the phytochrome chromophore, or phycocyanobilin, which may replace the phytochrome chromophore, resulted in the rescue of ptr116 phototropism. The ptr116 mutant and the phenotypically-related mutant ptr1 contain lower chlorophyll levels than the wild-type. Chlorophyll content of wildtype and mutant tissue grown under different light conditions was estimated using conventional spectrophotometry of extracts and fluorimetrically, on single apical cells. Dark-grown tissue contained about 100 times less chlorophyll than tissue grown under standard white light conditions. Red light given for 24 h to dark adapted filaments induced an increase in the chlorophyll content in the wildtype, but not in ptr116. Blue light induced an increase in chlorophyll both in wildtype and in ptr116. The red light effect on the wildtype was partially reversible with far-red. If ptr116 was grown on phycocyanobilin, an increase in chlorophyll was also found when cells were irradiated with red light. The results indicate that phytochrome as well as a blue light photoreceptor regulate chlorophyll accumulation in C. purpureus protonemata. It can be assumed that in ptr116, the synthesis of the phytochrome chromophore is blocked specifically beyond the synthesis common to chlorophyll and the phytochrome chromophore and affects an enzymatic step between protoporphyrin and biliverdin.     

Identification and characterization of the last two unknown genes,dapC and dapF,in the succinylase branch of the L-lysine biosynthesis of Corynebacterium glutamicum

The inspection of the complete genome sequence of Corynebacterium glutamicum ATCC 13032 led to the identification of dapC and dapF, the last two unknown genes of the succinylase branch of the L-lysine biosynthesis. The deduced DapF protein of C. glutamicum is characterized by a two-domain structure and a conserved diaminopimelate (DAP) epimerase signature. Overexpression of dapF resulted in an 8-fold increase of the specific epimerase activity. A defined deletion in the dapF gene led to a reduced growth of C. glutamicum in a medium with excess carbon but limited ammonium availability. The predicted DapC protein of C. glutamicum shared 29% identical amino acids with DapC from Bordetella pertussis, the only enzymatically characterized N-succinyl-aminoketopimelate aminotransferase. Overexpression of the dapC gene in C. glutamicum resulted in a 9-fold increase of the specific aminotransferase activity. A C. glutamicum mutant with deleted dapC showed normal growth characteristics with excess carbon and limited ammonium. Even a mutation of the two genes dapC and ddh, interrupting both branches of the split pathway, could be established in C. glutamicum. Overexpression of the dapF or the dapC gene in an industrial C. glutamicum strain resulted in an increased L-lysine production, indicating that both genes might be relevant targets for the development of improved production strains.     

A highly sensitive taste receptor cell for pyrrolizidine alkaloids in the lateral galeal sensillum of a polyphagous caterpillar, Estigmene acraea

Adult males of Estigmene acraea use pyrrolizidine alkaloids to produce pheromones and all stages probably use pyrrolizidine alkaloids for defense. The alkaloids are obtained from plants by the caterpillars. We demonstrate that a contact chemoreceptor neuron in the lateral galeal sensillum exhibits a dose-dependent response to seneciphylline N-oxide, a widely occurring pyrrolizidine alkaloid, down to concentrations of 10(-9) x mol l(-1), and even at 10(-12) x mol l(-1) the response is greater than to salt alone. At concentrations of 10(-6) mol x l(-1) and above the instantaneous firing rate is very high, and at 10(-4) mol x l(-1) initially exceeds 500 spikes s(-1). The firing rate declines in the 200 ms following stimulus onset but then is sustained with an instantaneous firing rate in excess of 100 spikes s(-1) for at least the next 800 ms. At lower concentrations a delay occurs before firing is initiated, and then the pattern of firing is irregular. The cell is equally sensitive to some but not all of several other pyrrolizidine alkaloids tested as free bases and their N-oxides. It also responds to ouabain, which may also serve as a defensive compound, and to asparagine and fructose but with much higher thresholds than to the pyrrolizidine alkaloids.     

Administration of keratinocyte growth factor down-regulates the pulmonary capacity of acetylcholine production

Keratinocyte growth factor protects the lung against various injurious stimuli. The protective mechanisms, however, are not yet fully understood. The aim of this study is to determine the influence of keratinocyte growth factor on the pulmonary capacity to synthesize acetylcholine, a potent regulator of pulmonary functions which is potentially involved in lung damage. Rats were treated twice (days 1 and 2) intratracheally with keratinocyte growth factor and analyzed at day 4. The mRNA expression of choline acetyltransferase - the acetylcholine synthesizing enzyme - was analyzed by real-time RT-PCR in the lung and in isolated alveolar epithelial type II cells. Choline acetyltransferase protein was assessed by immunoblotting and immunohistochemistry. Finally, pulmonary acetylcholine content was assessed biochemically. Keratinocyte growth factor-treatment led to decreased levels of choline acetyltransferase mRNA in the lung and in isolated alveolar epithelial type II cells. Accordingly, pulmonary choline acetyltransferase protein levels were reduced and pulmonary acetylcholine content declined from 2.8 nmol (control) to 0.4 nmol acetylcholine per gram of wet weight. In conclusion, the present data show that the potent regulator of pulmonary functions, acetylcholine, is produced by the major pulmonary target cell of keratinocyte growth factor, that is alveolar epithelial type II cells. Acetylcholine synthesis is down-regulated by keratinocyte growth factor administration which might contribute to lung protection and to harmonize surfactant homeostasis under conditions of keratinocyte growth factor-induced alveolar epithelial type II cell hyperplasia.     

Polymorphisms in the promoter region of ESR2 gene and breast cancer susceptibility

Genetic variations like single nucleotide polymorphisms (SNPs) in genes involved in estrogen biosynthesis, metabolism and signal transduction have been suggested to affect breast cancer susceptibility. In this study we tested the hypothesis that polymorphisms in the promoter of ESR2 gene may be associated with increased risk for breast cancer. We analyzed three SNPs in the promoter region of human ESR2 gene by means of allele-specific tetra-primer PCR. A total of 318 sporadic breast cancer cases and 318 age-matched controls were included in the study. With regard to homozygous genotypes, women with sporadic breast cancer more frequently carried the CC genotype of ESR2 promoter SNP rs2987983 (OR 1.99, p = 0.005). Calculation of allele positivity demonstrated that presence of T allele of this SNP was more frequent in healthy women. Our data suggest that a SNP in the promoter region of ESR2 gene might be able to affect breast cancer risk. These results further support the emerging hypothesis that ERβ is an important factor in breast cancer development.