Regionally Distinct N-Methyl-D-Aspartate Receptors Distinguished by Quantitative Autoradiography of [3H]MK-801 Binding in Rat Brain

Abstract: Quantitative autoradiography of [³H]MK-801 binding was used to characterize regional differences in N -methyl- d -aspartate (NMDA) receptor pharmacology in rat CNS. Regionally distinct populations of NMDA receptors were distinguished on the basis of regulation of [³H]MK-801 binding by the NMDA antagonist 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP). CPP inhibited [³H]MK-801 binding in outer cortex (OC) and medial cortex (MC) with apparent K _i values of 0.32-0.48 μ M , whereas in the medial striatum (MS), lateral striatum (LS), CA1, and dentate gyrus (DG) of hippocampus, apparent K _i values were 1.1-1.6 μ M . In medial thalamus (MT) and lateral thalamus (LT) the apparent K _i values were 0.78 μ M . In the presence of added glutamate (3 μ M ), the relative differences in apparent K _i values between regions maintained a similar relationship with the exception of the OC. Inhibition of [³H]MK-801 binding by the glycine site antagonist 7-chlorokynurenic acid (7-ClKyn) distinguished at least two populations of NMDA receptors that differed from populations defined by CPP displacement. 7-ClKyn inhibited [³H]MK-801 binding in OC, MC, MS, and LS with apparent K _i values of 6.3-8.6 μ M , whereas in CA1, DG, LT, and MT, K _i values were 11.4-13.6 μ M . In the presence of added glycine (1 μ M ), the relative differences in apparent K _i values were maintained. Under conditions of differential receptor activation, regional differences in NMDA receptor pharmacology can be detected using [³H]MK-801 binding.     

Glutamate Receptor-Mediated Calcium Surges in Neurons Derived from P19 Cells

Abstract: Retinoic acid-treated murine P19 embryonal carcinoma cells differentiate into cells with neuronal morphology that display typical neuronal markers. In this study, the presence of glutamate receptors linked to Ca²⁺-signaling mechanisms on these neurons was demonstrated by testing the effects of glutamate agonists and antagonists on the intracellular calcium ion concentration ([Ca²⁺]_i). Glutamate (1 m M ) induced either sustained or transient increases in [Ca²⁺]_i. The sustained glutamate-induced increase in [Ca²⁺]_i was mimicked by NMDA (40 µ M ). The NMDA-triggered [Ca²⁺]_i response was abolished by incubating the cells in Ca²⁺-free medium or by pretreating them with Mg²⁺ (2 m M ) or MK-801 (0.1 µ M ). These responses were unaffected by the non-NMDA antagonist CNQX (10 µ M ), but they required glycine (3–30 µ M ). Kainate (40 µ M ) and AMPA (40 µ M ) did not affect [Ca²⁺]_i. Without external Ca²⁺, glutamate triggered transient, sometimes oscillating, increases in [Ca²⁺]_i. These responses were mimicked by the metabotropic agonist trans -(1 S ,3 R )-1-amino-1,3-cyclopentanedicarboxylic acid (300 µ M ). These results suggest that neurons derived from P19 embryonal carcinoma cells have NMDA and metabotropic, but not AMPA/kainate receptors, which are linked to Ca²⁺-signaling mechanisms. These cells could provide a consistent and reproducible model with which to study neuronal differentiation, neurotoxicity, and glutamate receptor-signaling mechanisms.     

Genes for the major structural components of Thermotogales species' togas revealed by proteomic and evolutionary analyses of OmpA and OmpB homologs

The unifying structural characteristic of members of the bacterial order Thermotogales is their toga, an unusual cell envelope that includes a loose-fitting sheath around each cell. Only two toga-associated structural proteins have been purified and characterized in Thermotoga maritima: the anchor protein OmpA1 (or Ompα) and the porin OmpB (or Ompβ). The gene encoding OmpA1 (ompA1) was cloned and sequenced and later assigned to TM0477 in the genome sequence, but because no peptide sequence was available for OmpB, its gene (ompB) was not annotated. We identified six porin candidates in the genome sequence of T. maritima. Of these candidates, only one, encoded by TM0476, has all the characteristics reported for OmpB and characteristics expected of a porin including predominant β-sheet structure, a carboxy terminus porin anchoring motif, and a porin-specific amino acid composition. We highly enriched a toga fraction of cells for OmpB by sucrose gradient centrifugation and hydroxyapatite chromatography and analyzed it by LC/MS/MS. We found that the only porin candidate that it contained was the TM0476 product. This cell fraction also had β-sheet character as determined by circular dichroism, consistent with its enrichment for OmpB. We conclude that TM0476 encodes OmpB. A phylogenetic analysis of OmpB found orthologs encoded in syntenic locations in the genomes of all but two Thermotogales species. Those without orthologs have putative isofunctional genes in their place. Phylogenetic analyses of OmpA1 revealed that each species of the Thermotogales has one or two OmpA homologs. T. maritima has two OmpA homologs, encoded by ompA1 (TM0477) and ompA2 (TM1729), both of which were found in the toga protein-enriched cell extracts. These annotations of the genes encoding toga structural proteins will guide future examinations of the structure and function of this unusual lineage-defining cell sheath.     

Temperature responses are a window to the physiology of dark respiration: differences between CO2 release and O2 reduction shed light on energy conservation

We showed that temperature responses of dark respiration for foliage of Pinus radiata could be approximated by Arrhenius kinetics, whereby E ₀ determines shape of the exponential response and denotes overall activation energy of respiratory metabolism. Reproducible and predictable deviation from strict Arrhenius kinetics depended on foliage age, and differed between R _CO2 and R _O2. Inhibition of oxygen reduction ( R _O2) by cyanide (inhibiting COX) or SHAM (inhibiting AOX) resulted in reproducible changes of the temperature sensitivity for R _O2, but did not affect R _CO2. Enthalpic growth – preservation of electrons in anabolic products – could be approximated with knowledge of four variables: activation energies ( E ₀) for both R _CO2 and R _O2, and basal rates of respiration at a low reference temperature ( R _REF). Rates of enthalpic growth by P. radiata needles were large in spring due to differences between R _REF of oxidative decarboxylation and that of oxygen reduction, while overall activation energies for the two processes were similar. Later during needle development, enthalpic growth was dependent on differences between E ₀ for R _CO2 as compared with R _O2, and increased E ₀( R _O2) indicated greater contributions of cytochrome oxidase to accompany the switch from carbohydrate sink to source. Temperature-dependent increments in stored energy can be calculated as the difference between R _CO2▵ H _CO2 and R _O2▵ H _O2.     

Elevated oxygen uptake and high rates of nitrogen excretion in early life stages of the cobia Rachycentron canadum (L.), a fast-growing subtropical fish

Physiological energetics of cobia Rachycentron canadum were quantified for 18 to 82 days post-hatch (dph) hatchery-reared juveniles to better understand energy transformation and its implications in growth and survival. Mean oxygen consumption rates ( ; mg O₂ h⁻¹) of fish fed ad libitum and fish that were starved significantly increased with increasing wet mass (M; g), = 1·4291 M ^0·8119 and = 1·1784 M ^0·7833, respectively, with a significant reduction in mean metabolic rates of starved fish (19 to 27% specific dynamic action; SDA). Total ammonia nitrogen excretion rates ( A _MM, μmol h⁻¹) also scaled with M and significantly decreased after starvation. Mean mass-specific A _MM and urea excretion rates are the highest reported in the literature, with urea accounting for approximately half the total nitrogen excretion measured in both fed and starved fish. Relatively high energetic rates may allow cobia to develop rapidly into pre-juveniles and be less susceptible to predation and starvation at a comparatively early age.     

Production and characterisation of monoclonal antibodies to the principle sonicate antigens of Porphyromonas gingivalis w50

Abstract Protein antigens from whole cell sonicates of Porphyromonas gingivalis W50, previously shown to be discriminatory antigens for patients with adult periodontitis, were purified using SDS-PAGE. Electroeluted proteins were used to immunize mice for the production of monoclonal antibodies (mAbs). A combination of enzyme-linked immunosorbent assay (ELISA) and Western blotting were used to screen hybridoma supernatants for mAbs. MAbs were successfully raised against M _r 115 000, M _r 55 000 and M _r 47 000 antigens together with a second M _r 55 000 polypeptide which was a contaminant of the M _r 55 000 antigen. No immunological cross-reactivity was found between these four proteins. The mAbs were used to examine the distribution of these antigens among fifteen P. gingivalis strains together with related oral bacteria using immunostaining of dot blots and Western blots. The antigens were confined to P. gingivalis with the M _r 115 000 and M _r 47 000 antigens being present in all strains tested . The distribution of the M _r 55 000 antigens were slightly more restricted: one M _r 55 000 (outer membrane location) was present in nine of the fifteen P. gingivalis strains tested, while the other M _r 55 000 (location unknown) was only absent from one strain. Whole cell ELISA demonstrated that the M _r 115 000 and the outer membrane M _r 55 000 antigen possess epitopes which are located on the surface of the bacterium.     

The effects of MK-801, ifenprodil, JO 1784, JO 1994 and JO 1997 on PK 11195 receptor binding, nitric oxide synthase (no synthase) activity and infarct volume in a mouse model of focal cerebral ischaemia

Middle cerebral artery occlusion (MCAO) is a widely used surgical procedure for inducing focal cortical ischaemia in mice. In the present study, all experiments were performed on 4-week-old, male Swiss mice (OF-1 Iffa Credo, France), 20–25 g at the time of surgery. Sham-operated mice were subjected to simple exposure of the middle cerebral artery. Mice were injected with either MK-801, ifenprodil, JO 1784, JO 1994 or JO 1997 at the following time points after surgery; 5, 15, 45 min and 3, 6, 24, 30, 48 and 54 h. Mice were sacrificed 72 h after surgery and both ipsilateral and contralateral cortices were dissected in their entirely, weighed, and assayed for [³H]PK 11195 binding while the brain-stem and cerebellum were assayed for nitric oxide synthase (NO synthase) activity. In a separate experiment the area of ischaemic damage was determined planimetrically by means of an image analysis system. Coagulation of the middle cerebral artery induced a marked enhancement of the ipsilateral cortical ω₃ peripheral-type benzodiazepine binding site (PTBB'S) densities, an increase in NO synthase activity in the brain-stem and cerebellum, and an increase in the cortical infarct area. MK-801, ifenprodil, JO 1784, JO 1994 and JO 1997 demonstrated comparable neuroprotective effects on all three indices of cortical damage. A down-regulation of cortical ω₃ peripheral-type benzodiazepine binding site (PTBB'S) densities and a decrease in NOS activity occurred following pharmacological intervention. In contrast to JO 1784, JO 1994 and JO 1997 have a bimodal effect on ω₃ PTBB'S densities.     

Improved purification procedure and some serological and physica properties of cassava common mosaic virus from South America

Large quantities of cassava common mosaic virus (CCMV) were purified from systemically infected Nicotiana benthamiana plants. A polyclonal antiserum, with a titre of 1/128 in the tube precipitin test, was produced by immunising rabbits with purified virus. Viral antigens were detected in cassava, using both the double-antibody sandwich or plate-trapped antigen forms of enzyme-linked immunosorbent assay (ELISA). The virus reacted with antisera to the potexviruses potato virus X and tulip virus X in F(ab')₂ ELISA. As determined by ELISA, isolates of CCMV from cassava and chaya are closely serologically related to each other. Leaf extracts from infected N. benthamiana plants were infective to a dilution of 10^--⁴ but not 10^--⁵; after heating for 10 min at 65 °C but not 70 °C; and after storage at room temperature for 14 days. The virus has a sedimentation coefficient of 126 S_20,w, a single coat protein molecule of c . mol. wt 21 000, and a single-stranded RNA genome of c . mol. wt 2.0 ± 10⁶. Several dsRNA species, including the putative viral replicative form of c . mol. wt 4.1 ± 10⁶, were isolated from virus-infected cassava and N. benthamiana .     

Effects of feeding and induction strategy on the production of BmR1 antigen in recombinant E. coli

Aim:  To investigate the effects of feeding and induction strategies on the production of Bm R1 recombinant antigen.
Methods and Results:  Fed-batch fermentation was studied with respect to the specific growth rate and mode of induction to assess the growth potential of the bacteria in a bioreactor and to produce high yield of Bm R1 recombinant antigen. Cells were grown at a controlled specific growth rate (μ_set) during pre-induction, followed by constant feeding postinduction. The highest biomass (24·3 g l⁻¹) was obtained during fed-batch process operated at μ_set of 0·15 h⁻¹, whereby lower μ_set (0·075 h⁻¹) gave the highest protein production (9·82 mg l⁻¹). The yield of Bm R1 was increased by 1·2-fold upon induction with 1 mmol l⁻¹ IPTG (isopropyl-β- d -thiogalactoside) compared to using 5 mmol l⁻¹ and showed a further 3·5-fold increase when the culture was induced twice at the late log phase.
Conclusions:  Combination of feeding at a lower μ_set and twice induction with 1 mmol l⁻¹ IPTG yielded the best result of all variables tested, promising an improved method for Bm R1 production .
Significance and Impact of the Study:  This method can be used to increase the production scale of the Bm R1 recombinant antigen to meet the increasing demand for Brugia Rapid^™, a commercial diagnostic test for detection of brugian filariasis.     

Fumonisin B1 production by Fusarium proliferatum strains isolated from Allium fistulosum plants and seeds in Japan

Aims:  To test the fumonisin B₁ - producing ability of Fusarium proliferatum strains isolated from Welsh onion ( Allium fistulosum ) plants and seeds of commercial cultivars in Japan and to examine the applicability of PCR-based assays to discriminate between fumonisin B₁-producing and nonproducing isolates.
Methods and Results:  Fumonisin B₁ levels in 20 Fusarium isolates obtained from Welsh onion plants and seeds of seven commercial cultivars were determined by HPLC. Thirteen of the 20 isolates produced fumonisin B₁. PCR assay with FUM1 gene-specific primers amplified a DNA fragment (700 bp) only from fumonisin-producing isolates.
Conclusions:  Fusarium proliferatum isolates that can produce fumonisin B₁ were often associated with wilted Welsh onion plants and seeds of some commercial cultivars. The PCR assay with FUM1 gene-specific primers has the potential to discriminate between fumonisin B₁-producing and nonproducing isolates.
Significance and Impact of the Study:  This study revealed that F. proliferatum producing fumonisin B₁ is associated with Welsh onion plants and that commercial cultivar seeds may be contaminated with the fungus. PCR amplification of FUM1 gene can be a useful tool for the rapid identification of fumonisin B₁-producing F. proliferatum isolates.     

Phototrophic growth on n-fatty acids by species of the genus Rhodocyclus sensu Imhoff et al.

Abstract Type strains of Rhodocyclus purpureus, R. gelatinosus and R. tenuis along with three local isolates of R. gelatinosus were tested for growth in the light on n -fatty acids ranging in chain length from C₅ (valerate) to C₂₂ (docosanoate).
R. purpureus , the type species of the genus, was anomalous in its limited ability to grow on n -fatty acids; no fatty acids of chain length greater than C₉ (nonanoate) were utilized. R. gelatinosus and R. tenuis , on the other hand, utilized all fatty acids in the range C₅ to C₁₈ inclusive. R. gelatinosus showed some restricted ability to use C₂₀ (eicosanoate) and C₂₂ (docosanoate) fatty acids.     

A comparison of the fluorescent ELISA and antibiotic resistance identification techniques for use in ecological experiments with Rhizobium trifolii

The fluorescent ELISA technique for the identification of bacteria was compared with antibiotic resistant mutants as marker systems for use with Rhizobium trifolii in root nodules and in soil. With an effective(CP3B) and an ineffective (R4) strain as a mixed 1:1 inoculum, there was a highly significant correlation ( P < 0.001) between the two techniques when the plants were grown at pH 5.5 when the majority of nodules were inhabited by the effective strain. At pH 6.5, where the ineffective strain predominated in the nodules, there was no correlation. The reason was that 85% of R4 nodules had volumes less than 0.1 mm₃ with bacterial numbers obviously below the necessary threshold for detection using the serological method. Both methods were efficient at enumerating rhizobia from soils although the recovery rate from a brown earth soil was significantly higher than from a peat soil. Fluorescent ELISA was able to detect rhizobia at 8.0 times 10₅ cells/ml soil suspension (1 g soil to 10 ml water) in the brown earth soil and at 2.0 times 10₅ cells/ml in the peat soil. The results are discussed in terms of the limitations of both techniques in ecological studies.     

RAT BRAIN FOLATE IDENTIFICATION

Abstract— Endogenous rat brain folates were identified using column chromatography for folate separation, authentic folate standards, and microbiological assay. The total folate content of rat brain was 360 ng per gram brain (wet wt) and consisted of tetrahydropteroylpentaglutamate (H₄ PteGlu₅, 29%), H₄ PteGlu₆ (14%), H₄PteGlu₇ (7%), 5-CH₃-H₄PteGlu₄ (7%), 5-CH₃-H₄PteGlu₅ (13%), and small amounts of formyl-, methyl-, and H₄PteGlu_1-6 (~25%) and non-methylated folates having a glutamate chain over seven glutamates long (4%). Overall folate oxidation state and one carbon forms were H₄PteGlu_n (58%), 5-CH₃-H₄PteGlu_n (18%), 5- and 10-CHO-H₄PteGlu_n (16%), and H₂ PteGlu_n (8%). No PteGlu_n was detected.     

Heteromers of Glutamate Decarboxylase Isoforms Occur in Rat Cerebellum

Abstract: The subunit structure of brain glutamate decarboxylase in cerebellum was investigated by using gel electrophoresis and antisera that specifically recognize the individual isoforms of brain glutamate decarboxylase (termed GAD₆₅ and GAD₆₇). The antisera were prepared against peptides that corresponded to amino acid sequences specific to each isoform. Each antiserum reacted specifically with the appropriate peptide in an ELISA and with the appropriate form of GAD on immunoblots. Nondenaturing gradient gel electrophoresis indicated that GAD is principally multimeric with monomeric forms comprising <3% of the total. Immunoprecipitation and immunoaffinity chromatography experiments were performed with antisera W624 and W883, which were prepared against peptides specific to GAD₆₅ and GAD₆₇, respectively. Immunoprecipitates prepared from cerebellar supernatants with W624 contained both GAD₆₅ and GAD₆₇, whereas some GAD₆₇ was left in the supernatant. In a similar manner, immunoprecipitates prepared with W883 contained both GAD₆₅ and GAD₆₇, whereas some GAD₆₅ remained in the supernatant. In addition, immunoaffinity columns prepared with either W624 or W883 retained both GAD₆₅ and GAD₆₇ even after extensive washing. These results are consistent with the presence of heteromultimers of GAD₆₅ and GAD₆₇ in cerebellum in addition to homomers of each form.     

Contribution of current photosynthates to root respiration of non-nodulated Medicago sativa: effects of light and nitrogen supply

Abstract: The effects of light (PFD) and nitrogen (N) supply on root respiration of new C (currently assimilated carbon, R _new) and old C ( R _old) were analysed in non-nodulated Medicago sativa . Plants were pre-treated with high/low PFD and high/low N supply with a regular 16/8 h light/dark cycle. Five to eight weeks after planting current photosynthates were labelled with ¹³C and their contribution to root respiration was continuously measured during a 24 h day/night cycle. PFD conditions during labelling were either those of the pre-treatments (control, 25 or 6 mol m^-2 d^-1) or, for high PFD plants, 6 mol m^-2 d^-1 by shortening the photoperiod or reducing irradiance. The fraction of new C in the respiratory CO₂ increased during the light period, but remained constant in the dark period. In control plants, R _new contributed 40 % to the daily root respiration in high PFD/high N conditions. Continuously low PFD increased (50 %) and low N decreased (26 %) the contribution of R _new. Exposing plants from high PFD pre-treatments to a short photoperiod or to low PFD stimulated R _old, indicating mobilisation of reserve C. This stimulation was more pronounced in plants with high N supply than in those with low N supply. Comparison with other legumes suggested that R _new in root respiration was mainly defined by the ratio between the assimilatory capacity of the shoots and the maintenance costs of roots with a short-term capacity of buffering respiratory demand by mobilisation of reserves in situations of fluctuating PFD.     

Limitations of the enzyme-linked immunosorbent assay for routine determination of legume inoculant quality

Peat inoculants containing strains of either Rhizobium or Bradyrhizobium spp. were used to determine correlations between cell numbers and A₄₀₅ values obtained with double antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA) and indirect ELISA. ELISA values of inoculants containing strains of Rhizobium were weak and non-specific; with Bradyrhizobium spp. strains, readings were higher and cross-reactions negligible when heated inoculant suspensions were allowed to stand for 3 h before ELISA determinations were made. With soybean inoculant, correlation coefficients of r = 0.93 and 0.83 were obtained with DAS and indirect ELISA, respectively. A linear curve relating log cell numbers to A₄₀₅ values was used to determine the reliability of DAS ELISA values obtained over 2 years with tests on commercially produced soybean inoculants. In the range 5 times 10⁸-ca 3 times 10⁹ cells/g inoculant, DAS ELISA estimates closely followed plate counts but no significant correlation was found when inoculants contained >ca 3 times 10⁹ cells/g. With a minimum requirement of 1 times 10⁹ cells/g inoculant, discrepancies between DAS ELISA estimates and plate counts obtained with inoculants produced with gamma-irradiated peat would have resulted in the erroneous rejection or acceptance of 14.5% of all inoculants tested, based on DAS ELISA estimates. With inoculants produced with steam-sterilized peat, which was unfavourable for survival of strain WB1, 70.0% of the inoculants rejected because of low plate counts would have been acceptable on the basis of DAS ELISA estimates.     

OmpA‐mediated rickettsial adherence to and invasion of human endothelial cells is dependent upon interaction with α2β1 integrin

Rickettsia conorii, a member of the spotted fever group (SFG) of the genus Rickettsia and causative agent of Mediterranean spotted fever, is an obligate intracellular pathogen capable of infecting various mammalian cell types. SFG rickettsiae express two major immunodominant s urface c ell a ntigen (Sca) proteins, OmpB (Sca5) and OmpA (Sca0). While OmpB‐mediated entry has been characterized, the contribution of OmpA has not been well defined. Here we show OmpA expression in Escherichia coli is sufficient to mediate adherence to and invasion of non‐phagocytic human endothelial cells. A recombinant soluble C‐terminal OmpA protein domain (954–1735) with predicted structural homology to the Bordetella pertussis pertactin protein binds mammalian cells and perturbs R. conorii invasion by interacting with several mammalian proteins including β1 integrin. Using functional blocking antibodies, small interfering RNA transfection, and mouse embryonic fibroblast cell lines, we illustrate the contribution of α2β1 integrin as a mammalian ligand involved in R. conorii invasion of primary endothelial cells. We further demonstrate that OmpA‐mediated attachment to mammalian cells is in part dependent on a conserved non‐continuous RGD motif present in a predicted C‐terminal ‘pertactin’ domain in OmpA.Our results demonstrate that multiple adhesin–receptor pairs are sufficient in mediating efficient bacterial invasion of R. conorii.     

Effects of JO 1784, A selective sigma ligand, on the autoradiographic localization of M2 and M2 muscarinic receptor subtypes in trimethyltin treated rats

The distribution patterns of M₁ and M₂ muscarinic receptor subtypes following TMT and JO 1784 administration in the male Sprague-Dawley rat were investigated. In the present study, JO 1784 was injected in doses of 1, 4 and 16 mg/kg i.p. for one week prior to the single injection of TMT (8 mg/kg i.p.) and subsequently for 33 days. The effects of JO 1784 on the density of muscarinic receptor sub-types (M₁ and M₂) in the control and trimethyltin (TMT) treated rats were then evaluated. The topographic distribution and changes in muscarinic (M₁ and M₂) receptor densities were determined by means of autoradiography using [³H]quinuclidinylbenzilate (QNB). Both sub-types of muscarinic receptors contributed to the observed decrease in total muscarinic receptor binding in TMT-treated rats. In control rats, JO 1784 alone decreased M₁ receptor density in the amygdaloid nuclei, basal ganglia, cortex and hippocampus and decreased M₂ receptor density in the amygdaloid nuclei, basal ganglia, cortex, hippocampus, hypothalamus and septal regions. In TMT treated rats, chronic JO 1784 administration has a “neuroprotective effect” on both M₁ and M₂ receptors subtypes. Thus, following chronic administration of JO 1784 to TMT treated rats, both increases and decreases in M₁ receptor density were observed relative to TMT animals. A significant increase in M₁ receptor density was found in the cortex, olfactory regions, septum, thalamus and basal forebrain nuclei. In the hippocampus (CA₂ and CA₃), a significant decrease in M₁ receptor density was observed. In TMT-treated rats, JO 1784 produced a significant increase in M₂ receptor density in several brain regions with the most marked effects occurring in the amygdaloid nuclei, basal ganglia, cortex, hippocampus and hypothalamus. The ability of the selective sigma ligand, JO 1784, to attenuate the loss of muscarinic receptors in TMT treated rats could be of importance in the development of novel neuroprotective drugs.     

Effects of temperature, body size and feeding on rates of metabolism in young‐of‐the‐year haddock

The mean rate of oxygen consumption (routine respiration rate, R _R, mg O₂ fish⁻¹ h⁻¹), measured for individual or small groups of haddock Melanogrammus aeglefinus (3–12 cm standard length, L _S) maintained for 5 days within flow‐through respiratory chambers at four different temperatures, increased with increasing dry mass ( M _D). The relationship between R _R and M _D was allometric ( R _R = α  M ^b ) with b values of 0·631, 0·606, 0·655 and 0·650 at 5·0, 8·0, 12·0 and 15·0° C, respectively. The effect of temperature ( T ) and M _D on mean R _R was described by     indicating a Q ₁₀ of 2·27 between 5 and 15° C. Juvenile haddock routine metabolic scope, calculated as the ratio of the mean of highest and lowest deciles of R _R measured in each chamber, significantly decreased with temperature such that the routine scope at 15° C was half that at 5° C. The cost of feeding ( R _SDA) was c . 3% of consumed food energy, a value half that found for larger gadoid juveniles and adults.     

Dopamine-Releasing Action of 6R-l-erythro-Tetrahydrobiopterin: Analysis of Its Action Site Using Sepiapterin

Abstract: Recently, we reported that 6 R - l - erythro -tetrahydrobiopterin (6 R -BH₄), a natural cofactor for hydroxylases of tyrosine and tryptophan, has a monoamine-releasing action independent of its cofactor activity. Here we attempted to determine whether 6 R -BH₄ acts inside the cell or from the outside of the cell by using brain microdialysis in the rat striatum. For this purpose, sepiapterin, an immediate precursor of 6 R -BH₄ in the salvage pathway, was used to selectively increase the intracellular 6 R -BH₄ levels. Dialytic perfusion of sepiapterin increased tissue levels of reduced biopterin (mainly 6 R -BH₄) but not the extracellular levels. Administration of sepiapterin increased the extracellular levels of 3,4-dihydroxyphenylalanine (DOPA) (an index of in vivo tyrosine hydroxylase activity) and of dopamine (DA) (an index of in vivo DA release). Either of the increases was eliminated after pretreatment with a tyrosine hydroxylase inhibitor α-methyl- p -tyrosine. Administration of 6 R -BH₄ increased extracellular levels of reduced biopterin, DOPA, and DA. After pretreatment with α-methyl- p -tyrosine, the increase in DOPA levels was abolished, but most of the increase in DA levels persisted. The increase in DA levels also persisted after pretreatment with nitric oxide synthase inhibitors. These data demonstrate that 6 R -BH₄ stimulates DA release directly, independent of its cofactor action for tyrosine hydroxylase and nitric oxide synthase, by acting from the outside of neurons.