The physiological role of kainate receptors in the amygdala

The kainate subtype of glutamate receptors has received considerable attention in recent years, and a wealth of knowledge has been obtained regarding the function of these receptors. Kainate receptors have been shown to mediate synaptic transmission in some brain regions, modulate presynaptic release of glutamate and gamma-aminobutyric acid (GABA), and mediate synaptic plasticity or the development of seizure activity. This article focuses on the function of kainate receptors in the amygdala, a brain region that plays a central role in emotional behavior and certain psychiatric illnesses. Evidence is reviewed indicating that postsynaptic kainate receptors containing the glutamate receptor 5 kainate receptor (GLUk5) subunit are present on interneurons and pyramidal cells in the basolateral amygdala and mediate a component of the synaptic responses of these neurons to glutamatergic input. In addition, GLUk5-containing kainate receptors are present on presynaptic terminals of GABAergic neurons, where they modulate the release of GABA in an agonist concentration-dependent, bidirectional manner. GLUk5-containing kainate receptors also mediate a longlasting synaptic facilitation induced by low-frequency stimulation in the external capsule to the basolateral nucleus pathway, and they appear to be partly responsible for the susceptibility of the amygdala to epileptogenesis. Taken together, these findings have suggested a prominent role of GLUk5-containing kainate receptors in the regulation of neuronal excitability in the amygdala.     

Monitoring of ozone pollution and the physiological activity of <Emphasis Type="Italic">Pinus halepensis</Emphasis> (Mill.) by electron paramagnetic resonance and other parameters

An investigation of selected Aleppo pines in the forests of Mt Hymettus and Mt Parnis near Athens (Greece) was undertaken at three different sites in the period 1999–2003, because a considerable proportion of pine trees showed visible signs of chlorotic mottle. This condition is characteristic of high and prolonged levels of ozone exposure. Needles from Aleppo pine trees (Pinus halepensis Mill.) were analyzed for their manganese content in combination with Electron Paramagnetic Resonance (EPR) spectra of Mn²⁺, involved in photosystem II. Manganese is considered as an important bioindicator for the vitality of trees. Also, we investigated the EPR spectrum of the needles in the region of g=2.0045 for healthy and diseased trees. The antioxidant capacity of the needles extract was measured from trees by the DPPH method. Finally, seasonal changes in chlorophyll concentration in the needles were measured to evaluate the effects of ozone. Measurements of ozone concentrations at the three sites showed that there were elevated levels during the summer months. Our experimental results suggest that the concentration of manganese in the needles was lower in the area with higher ozone concentrations, supported by EPR measurements. Higher ozone concentrations also affected the antioxidant potential of the needles and their chlorophyll content during summer months. Our findings also confirmed the resilience of Aleppo pines under stressful conditions and recovery in winter months. Despite the experimental problems, EPR spectra of Mn²⁺ in combination with other methods can be used as a sensitive bioindicator for ozone pollution, and is the result of oxidative stress affecting the growth cycle of the pine trees and their photosynthetic mechanisms.     

Kinetics of methyl t-butyl ether cometabolism at low concentrations by pure cultures of butane-degrading bacteria

Butane-oxidizing Arthrobacter (ATCC 27778) bacteria were shown to degrade low concentrations of methyl t-butyl ether (MTBE; range, 100 to 800 microg/liter) with an apparent half-saturation concentration (K(s)) of 2.14 mg/liter and a maximum substrate utilization rate (k(c)) of 0.43 mg/mg of total suspended solids per day. Arthrobacter bacteria demonstrated MTBE degradation activity when grown on butane but not when grown on glucose, butanol, or tryptose phosphate broth. The presence of butane, tert-butyl alcohol, or acetylene had a negative impact on the MTBE degradation rate. Neither Methylosinus trichosporium OB3b nor Streptomyces griseus was able to cometabolize MTBE.     

Isolation of high-affinity ligand-binding proteins by periplasmic expression with cytometric screening (PECS)

Periplasmic expression with cytometric screening (PECS) is a powerful and rapid "display-less" technology for isolating ligand-binding proteins from diverse libraries. Escherichia coli expressing a library of proteins secreted into the periplasmic space are incubated with a fluorescent conjugate of the target ligand. Under the proper conditions, ligands as large as about 10 kDa can equilibrate within the periplasmic space without compromising the cell's integrity or viability. The bacterial cell envelope effectively serves as a dialysis bag to selectively retain receptor-fluorescent probe complexes but not free ligand. Cells displaying increased fluorescence are then isolated by flow cytometry. We demonstrate that scFv antibodies with both very high and low affinity to digoxigenin can be isolated from libraries screened by PECS using a benchtop flow cytometer. We also show that preexisting libraries constructed for display on filamentous bacteriophage can be screened by PECS without the need for subcloning. In fact, PECS was found to select for proteins that could be missed by conventional phage panning and screening methods.     

Molecular Characterization of the Host-Adapted Pathogen Verticillium longisporum on the Basis of a Group-I Intron Found in the Nuclear SSU-rRNA Gene

Verticillium wilt of oilseed rape is caused by the host-adapted pathogen Verticillium longisporum comb. nov. With one set of nuclear SSU-rRNA gene primers, a PCR amplification product of ca. 2.5 kb was generated from all isolates of V. longisporum tested (36 from Europe, Japan, and USA), with the exception of two recombinant isolates. On the contrary, all the other phytopathogenic and non-phytopathogenic species of Verticillium tested (18 species, 46 isolates), with the exception of one isolate of V. lecanii and two of Cordyceps sp., generated a product of ca. 1.65 kb. Sequence analysis of the SSU-rRNA gene of two typical isolates of V. longisporum (wild radish, Japan, and oilseed rape, Germany) revealed that this dimorphism was due to the presence of an identical 839-bp intron located in a highly conserved insertion position (nt 1165 of Saccharomyces cerevisiae). The intron sequence was classified as group-I intron on the basis of conserved sequence and secondary structural elements. Primers designed from the 839-bp intron sequence amplified only the V. longisporum. Phylogenetic analysis based on SSU-rDNA sequences showed that V. longisporum was closely related to the genera of other filamentous Ascomycetes with fruiting bodies. Received: 24 August 2000 / Accepted: 25 September 2000     

A hollow-fiber membrane bioreactor for the removal of trichloroethylene from the vapor phase

A hollow-fiber membrane bioreactor was used to separate trichloroethylene (TCE) from a gaseous waste stream with subsequent cometabolic biodegradation by a pure culture of Methylosinus trichosporium OB3b PP358. The two-stage bioreactor system was successfully operated for 20 days. PP358 was grown in a continuous-flow chemostat and circulated through the fiber lumen of a hollow-fiber membrane module (HFMM), while TCE contaminated air (141 to 191 microg/L) was pumped through the HFMM shell. Between 54% -84% TCE transfer and 92%-96% TCE cometabolism were obtained in the HFMM reactor loop. Short shell-residence times, 1.6 to 5.0 minutes, demonstrated quick throughput of TCE contaminated air. Best-fit computer modeling of the biological experiments estimated mass transfer coefficients between 2.0 x 10(-3) cm/min and 5.6 x 10(-3) cm/min. The average pseudo-first-order biodegradation rate constant for the biological experiments was 0.46 L/mg TSS/d. These results demonstrate that the hollow-fiber membrane bioreactor represents an attractive technology for the bioremediation of gaseous waste streams.     

Sandhoff disease in Cyprus: population screening by biochemical and DNA analysis indicates a high frequency of carriers in the Maronite community

In the last 15 years, four patients with the infantile form of Sandhoff disease were diagnosed in four different families in Cyprus (population 703,000, birth rate 1.7%). Three of these cases came from the Christian Maronite community (less than 1% of the population) and one from the Greek community (84% of the population). This relatively large number of patients prompted us to initiate an epidemiological study in order to establish the frequency of the mutant allele in Cyprus. Carrier detection was initially based on the measurement of beta-hexosaminidase A and B in both leucocytes and serum. Using the enzyme test, 35 carriers were identified among 244 random Maronite samples and 15 among 28 Maronites with a family history of Sandhoff disease, but only one carrier was found out of 115 random samples from the Greek community. In parallel to the biochemical screening, DNA studies were undertaken in one of the three Maronite patients and in a Greek carrier related to the Greek patient. These studies resulted in the identification of two novel mutations, a deletion of A at nt76 and a G to C transversion at position 5 of the 5'-splice site of intron 8, which have been published. We subsequently screened the carriers detected in the biochemical study for these two mutations using PCR-based tests. Of 50 Maronite carriers examined, 42 were found to have the nt76 deletion. Eight Maronite samples, designated carriers from the biochemical results, were negative for both mutations. It is possible that these individuals were incorrectly classified as carriers since their enzyme values are equivocal, although the presence of another mutation has not been excluded. Two Greek Cypriot carriers and two obligate Lebanese carriers were negative for both mutations. We conclude that there is a high frequency of Sandhoff disease carriers in the Maronite community of Cyprus, approximately 1 in 7, and that a single mutation predominates in this population.     

Expression profile and interactions of hnRNP A3 within hnRNP/mRNP complexes in mammals

The hnRNP A/B family contains abundant nuclear proteins with major roles in alternative splicing and the ability for nucleo-cytoplasmic shuttling. Compared to the best known members of this family (hnRNP A1, A2/B1), hnRNP A3 is a relatively less known protein. We report herein immunochemical studies with the hnRNP A3 isoforms (A3a and A3b) that provided evidence for species-specific expression. The unspliced A3a was found in human and murine cells, while the spliced A3b was a unique and abundant isoform in mouse/rat. In addition, a tissue-specific variation was observed in mice, as the brain was the only tissue found to overexpress hnRNP A3a. Both hnRNP A3a and A3b were able to stably associate with immunoselected hnRNP and mRNP complexes. Use of the auxiliary domain of hnRNP A3 in pull-down assays on human cell extracts revealed its unique ability to form a network of interactions not only with other A/B proteins but also with additional hnRNPs. All interactions, except those of hnRNP A1, were highly enhanced by previous RNase A digestion of the extracts. Our findings revealed novel characteristics of hnRNP A3 and supported its extensive involvement in the many aspects of mRNA maturation processes along with the other hnRNP A/B proteins.