μ-Opioid Receptors Mediate the Inhibitory Effect of Opioids on Dopamine-Sensitive Adenylate Cyclase in Primary Cultures of Rat Neostriatal Neurons

The receptors mediating the inhibition of D1 dopamine receptor-stimulated adenylate cyclase by opioids were examined in primary cultures of rat neostriatal neurons. Adenylate cyclase activity was dose-dependently increased by the selective D1 dopamine receptor agonist SKF 38393 (EC50 = 0.05 microM). This stimulation was fully antagonized by the selective D1 dopamine receptor antagonist SCH 23390 (1 microM). SKF 38393 (1 microM)-stimulated adenylate cyclase activity was strongly reduced (by almost 60%) by the highly selective mu-agonist [D-Ala2, MePhe4, Gly-ol5]-enkephalin (DAGO; EC50 = 0.006 microM) and high concentrations of the selective delta-agonist [D-Ser2(O-tert-butyl), Leu5]-enkephalyl-Thr6 (DSTBU-LET; EC50 = 0.13 microM) but not by the selective delta-agonist [D-penicillamine2, D-penicillamine5]enkephalin (DPDPE). D1 dopamine receptor-stimulated adenylate cyclase activity was also slightly reduced (by approximately 20%) by high concentrations of the kappa-agonist U50,488 (EC50 = 0.63 microM). The inhibitory effects of submaximally effective concentrations of DAGO, DSTBULET, and U50,488 were equally well antagonized by the mu-opioid receptor-selective antagonist naloxone (EC50 of approximately 0.1 microM). Neither the irreversible delta-ligand fentanyl isothiocyanate (1 microM) nor the reversible delta-antagonist ICI 174864 (1 microM) reversed the inhibitory effects of DSTBULET. The inhibitory effects of DAGO and U50,488 were equally well reversed by high concentrations (greater than 0.1 microM) of the kappa-opioid receptor-selective antagonist norbinaltorphimine. The effect of DAGO (1 microM) was already detectable after 1 day in culture, whereas DPDPE (1 microM) had no effect even after 28 days in culture. These data indicate that an homogeneous population of mu-opioid receptors coupled as inhibitors to D1 dopamine receptor-stimulated adenylate cyclase is expressed in rat neostriatal neurons in primary culture.     

Linker insertion mutagenesis of herpesviruses: inactivation of single genes within the Us region of pseudorabies virus.

We describe a technique for the systematic inactivation of nonessential genes within the genome of a herpesvirus without the requirement for phenotypic selection. This technique is based on the insertion of an oligonucleotide containing translational stop codons at a random site within a large cloned viral DNA fragment. Mutant virus is then reconstituted by cotransfection with overlapping viral clones, together comprising the entire viral genome, as described previously (M. van Zijl, W. Quint, J. Briaire, T. de Rover, A. Gielkens, and A. Berns, J. Virol. 62:2191-2195, 1988). This technique was used to construct, in a single experiment, a set of 13 viable pseudorabies virus strains with oligonucleotide insertions within all known genes of the Us region except for the gp50 gene, which proved essential for virus growth in cell culture. The growth rate in porcine kidney cells of mutants of all nonessential Us genes was similar to that of the parental virus, with the exception of a mutant of the recently identified protein kinase gene.     

Ethanol increases PGE and thromboxane production in mouse pregnant uterine tissue

The teratogenic effect of ethanol in the C57BL/6J mouse can be attenuated by pretreatment with aspirin (ASA). One prominent effect of ASA is to inhibit prostaglandin (PGE) and thromboxane (TXB2) production. We examined the effect of in vivo ethanol exposure on PGE and TXB2 production in a uterine-embryo tissue sample of C57BL/6J mice either before or after in vivo ASA pretreatment on day 10 of gestation. Ethanol increased both PGE and TXB2 production by approximately 20%. ASA caused a marked reduction of PGE and TXB2 in both control and ethanol groups by approximately 80-90%. The mouse strain, gestation time, and study parameters used in this study were the same as in the previously reported ASA attenuation of the teratogenic effect of ethanol. Therefore, the present data add additional support to the hypothesis that prostaglandin and/or thromboxane production may be involved in at least some aspects of fetal alcohol syndrome.     

Demonstration of sensory-motor innervation of postmetamorphic forelimb regenerates of Triturus alpestris (Urodela) on cryotome serial sections using horseradish peroxidase

A method has been developed to obtain horseradish peroxidase-treated serial sections containing spinal cord as well as bilateral ventral and dorsal roots, dorsal root ganglia and spinal nerves. Young postmetamorphic newts (Triturus alpestris) served as experimental animals. After cryotome cross sectioning the forelimb region of the trunk, slices 80 microns in thickness were mounted serially with up to 15 sections per slide. This facilitated subsequent staining manipulations and made partial loss of sections less likely.     

Fine needle aspiration of metastatic and hematologic malignancies clinically mimicking pancreatic carcinoma.

The fine needle aspiration (FNA) cytology findings in 19 cases of hematopoietic and metastatic neoplasms that radiographically mimicked primary pancreatic carcinoma are reported. These cases represented 11% of 176 malignant diagnoses in a series of 304 pancreatic FNAs. The cytologic diagnoses included 7 non-Hodgkin's lymphomas, 2 Hodgkin's lymphomas, 6 small cell carcinomas (4 lung, 1 gallbladder, 1 skin), 3 squamous cell carcinomas (2 cervix, 1 esophagus) and 1 hepatocellular carcinoma. In six cases the pancreatic lesion was the initial presentation of malignant disease. These included five lymphomas, which probably involved peripancreatic lymph nodes, and a metastatic small cell carcinoma of pulmonary origin. Recognition of unusual morphologic features of pancreatic carcinoma raised the possibility of extrapancreatic malignancies. Electron microscopy and immunocytochemistry performed on FNA specimens were helpful in selected cases. The FNA diagnosis of hematopoietic and metastatic neoplasms that clinically mimic pancreatic carcinoma prompts appropriate clinical studies and treatment and eliminates the need for open pancreatic biopsy and/or resection.     

Expression of active spinach glycolate oxidase in Aspergillus nidulans

The biocatalytic production of glyoxylic acid from glycolic acid requires two enzymes: glycolate oxidase, which catalyzes the oxidation of glycolic acid by oxygen to produce glyoxylic acid and hydrogen peroxide, and catalase, which decomposes the byproduct hydrogen peroxide. As an alternative to isolation from the leaf peroxisomes of spinach, glycolate oxidase has now been cloned and expressed in transformants of Aspergillus nidulans T580 at levels ranging from 1.7 to 36 IU/g dry wt. cells. The glycolate oxidase of transformant strain T17 comprises ca. 1.9% of total cell protein and is expressed at near 100% activity. (c) 1996 John Wiley & Sons, Inc.     

Thermodynamics elucidation of the structural stability of a thermophilic protein

The structural stability of the protein, phycocyanin isolated from two strains of cyanophyta, Synechococcus lividus (thermophile) and Phormidium luridum (mesophile), are investigated by comparative thermal and denaturant unfolding, using differential scanning calorimetry, visible absorption spectrophotometry, and circular dichroism. The thermophilic protein exhibits a much higher temperature and enthalpy of unfolding from the native to the denatured state. The concentration of urea at half-completion of thermal unfolding is essentially the same between the thermophilic and mesophilic proteins; in contrast, the corresponding temperature and the enthalpy of thermal unfolding are much higher for the thermophilic protein. In addition, the concentration of urea at which the non-thermal (denaturant) unfolding of protein is half-completed, as detected by either circular dichroism or absorption spectroscopy, is significantly higher in the thermophilic protein, while the apparent free energy of unfolding only shows a moderate difference between the two proteins. The distinct differences in the enthalpy of thermal unfolding and the free energy of denaturant unfolding are interpreted in terms of a significant entropy change associated with the unfolding of these proteins. This entropy contribution is much higher in the thermophilic protein, and may be derived from its more rigid overall structure that possesses higher internal hydrophobicity and stronger internal packing.     

Chemotype pattern differentiation of Thymus pulegioides on different substrates

Relationships between soil chemistry and population chemotype structure of Thymus pulegioides have been studied. The analysis of correlations suggest that an increased carbonate content in soil decreases the chemotype diversity of a population (as calculated by use of the Shannon index): the proportion of linalool chemotype plants rises and that of the phenol chemotype plants declines. In addition, the chemotype diversity decreases with increasing frequency of linalool chemotype, and increases with increase of carvacrol chemotype.     

Lignin-Degrading Enzymes of the Commercial Button Mushroom, Agaricus bisporus

Agaricus bisporus, grown under standard composting conditions, was evaluated for its ability to produce lignin-degrading peroxidases, which have been shown to have an integral role in lignin degradation by wood-rotting fungi. The activity of manganese peroxidase was monitored throughout the production cycle of the fungus, from the time of colonization of the compost through the development of fruit bodies. Characterization of the enzyme was done with a crude compost extract. Manganese peroxidase was found to have a pI of 3.5 and a pH optimum of 5.4 to 5.5, with maximal activity during the initial stages of fruiting (pin stage). The activity declined considerably with fruit body maturation (first break). This apparent developmentally regulated pattern parallels that observed for laccase activity and for degradation of radiolabeled lignin and synthetic lignins by A. bisporus. Lignin peroxidase activity was not detected in the compost extracts. The correlation between the activities of manganese peroxidase and laccase and the degradation of lignin in A. bisporus suggests significant roles for these two enzymes in lignin degradation by this fungus.