High affinity dopamine D2 receptor radioligands. 2. [125I]epidepride, a potent and specific radioligand for the characterization of striatal and extrastriatal dopamine D2 receptors.

Epidepride, (S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-iodo-2,3-dimethoxybenzamide+ ++, the iodine analogue of isoremoxipride (FLB 457), was found to be a very potent dopamine D2 receptor antagonist. Optimal in vitro binding required incubation at 25 degrees C for 4 h at pH 7.4 in a buffer containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2. Scatchard analysis of in vitro binding to striatal, medial frontal cortical, hippocampal and cerebellar membranes revealed a KD of 24 pM in all regions, with Bmax's of 36.7, 1.04, 0.85, and 0.37 pmol/g tissue, respectively. The Hill coefficients ranged from 0.91-1.00 in all four regions. The IC50's for inhibition of [125I]epidepride binding to striatal, medial frontal cortical, and hippocampal membranes for SCH 23390, SKF 83566, serotonin, ketanserin, mianserin, naloxone, QNB, prasozin, clonidine, alprenolol, and norepinephrine ranged from 1 microM to greater than 10 microM. Partial displacement of [125I]epidepride by nanomolar concentrations of clonidine was noted in the frontal cortex and hippocampus, but not in the striatum. Scatchard analysis of epidepride binding to alpha 2 noradrenergic receptors in the frontal cortex and hippocampus revealed an apparent KD of 9 nM. At an epidepride concentration equal to the KD for the D2 receptor, i.e. 25 pM, no striatal alpha 2 binding was seen and only 7% of the specific epidepride binding in the cortex or hippocampus was due to binding at the alpha 2 site. Correlation of inhibition of [3H]spiperone and [125I]epidepride binding to striatal membranes by a variety of D2 ligands revealed a correlation coefficient of 0.99, indicating that epidepride labels a D2 site. In vitro autoradiography revealed high densities of receptor binding in layers V and VI of prefrontal and cingulate cortices as well as in striatum. In vivo rat brain uptake revealed a hippocampal:cerebellar and frontal cortical:cerebellar ratio of 2.2:1 which fell to 1.1:1 following haloperidol pretreatment. These properties suggest that [125I]epidepride is a superior radioligand for the in vitro and in vivo study of striatal and extrastriatal dopamine D2 receptors.     

Downregulation of cell-to-cell communication by the viralsrc gene is blocked by TMB-8 and recovery of communication is blocked by vanadate

Summary The viralsrc gene downregulates junctional communication, closing cell-to-cell membrane channels presumably by way of the phosphoinositide signal route. We show that TMB-8 [8-N, N-(diethylamino) octyl-3,4,5-trimethoxybenzoate] counteracts this downregulation in cells transformed by temperature-sensitive mutant Rous sarcoma virus: TMB-8 (36–72 m) raises junctional permeability when applied during activity ofsrc protein kinase, i.e., at steady permissive temperature; and TMB-8 inhibits the fall of junctional permeability, when the activity ofsrc protein kinase gets turned on. TMB-8 also (reversibly) inhibits the growth of the cells at permissive temperature and reverses the morphological changes associated with transformation. The morphological reversal lags several hours behind the junctional-permeability reversal. Communication recovers within a few minutes when the activity of thesrc protein kinase is turned off (in absence of TMB-8). Sodium orthovanadate (20 m) prevents this recovery, but it has no major effect on junctional permeability on its own. We discuss possible modes of action of these agents on critical stages of the signal route, related to intracellular Ca²⁺ and protein kinase C.     

Development and fate of electron-dense microbodies in trap cells of the nematophagous fungusArthrobotrys oligospora

The development of electron-dense microbodies in cells of capture organs of the nematophagous fungus Arthrobotrys oligospora was studied with different ultrastructural techniques. Kinetic experiments revealed that the synthesis of these microbodies started in a very early stage of trap formation; the organelles originated from special regions of endoplasmic reticulum by budding. Mature organelles were surrounded by a single membrane of approximately 9 nm (KMnO4-fixation) and lacked crystalline inclusions. The presence of the electron-dense microbodies was independent of the conditions during which the traps had developed. The organelles remained intact during aging of the trap cells. They were also observed in the trophic hyphae after capture and penetration of nematodes. However, the distribution patterns of these organelles in the trophic hyphae, which were identical to those observed after germination of isolated traps on different cultivation media, suggested that their presence must be explained by dilution of organelles in newly formed cells.     

High Performance Liquid Chromatography of Molecular Species from Free Sterols and Sterylglycosides Isolated from Oat Leaves and Seeds

Free sterols and sterylglycosides (SG) from oat leaves and seedswere isolated by conventional thin layer chromatography (TLC)and subjected to high performance liquid chromatography (HPLC)for resolution of molecular species. Acylsterylglycosides, isolatedby TLC, were converted to SG by mild alkaline hydrolysis anddetermined as SG. Sterols and SG were injected onto the columnwithout any chemical treatment and the separated species weredetected at 200 nm. The separation of SG-species follows exactlythe separation of free sterols. Though gas liquid chromatography still is the method of choice,advantages of HPLC is to analyse directly the SG-species withouthydrolysis and derivatization as compared to GLC. After TLCthe sterol- and the SG-fraction are injected directly onto thecolumn. This is extremely important for labile sterylglycosidesor sterols, as demonstrated for the avenasterols. ¹ Preliminary reports have been presented on the "4. Arbeitstagung,Pflanzliche Lipide", October 7–8, 1983 in M?nster (FRG)and on the "6th International Symposium on the Structure, Functionand Metabolism of Plant Lipids", Neuchatel, Switzerland, July16–20, 1984. (Received November 12, 1984; Accepted January 14, 1985)     

Management of a harem breeding colony of rhesus monkeys to reduce trauma-related morbidity and mortality

A management procedure was developed for a harem breeding colony of rhesus monkeys to reduce trauma-related injuries and deaths resulting from the periodic removal of pregnant monkeys for research and their subsequent return to the population. Lower morbidity and mortality rates, a reduced mean conception interval, and a higher mean conception rate occurred when monkeys were maintained in permanent harems to which returning females were reintroduced compared to new social groups formed from aggregates of unfamiliar animals.     

Recognition sequences of restriction endonucleases and methylases--a review

The properties and sources of all known endonucleases and methylases acting site-specifically on DNA are listed. The enzymes are crossindexed (Table I), classified according to homologies within their recognition sequences (Table II), and characterized within Table II by the cleavage and methylation positions, the number of recognition sites on the DNA of the bacteriophages lambda, phi X174 and M13mp7, the viruses Ad2 and SV40, the plasmids pBR322 and pBR328 and the microorganisms from which they originate. Other tabulated properties of the restriction endonucleases include relaxed specificities (Table III), the structure of the restriction fragment ends (Table IV), and the sensitivity to different kinds of DNA methylation (Table V). Table VI classifies the methylases according to the nature of the methylated base(s) within their recognition sequences. This table also comprises those restriction endonucleases, which are known to be inhibited by the modified nucleotides. Furthermore, this review includes a restriction map of bacteriophage lambda DNA based on sequence data. Table VII lists the exact nucleotide positions of the cleavage sites, the length of the generated fragments ordered according to size, and the effects of the Escherichia coli dam- and dcmI-coded methylases M X Eco dam and M X Eco dcmI on the particular recognition sites.     

Upper limits of temperature for growth inChlorella (Chlorophyceae)

The upper limit of temperature for growth is a species-specific character in the genusChlorella. The limits of 14Chlorella species range from 26–30°C (C. saccharophila) to 38–42°C (C. sorokiniana), withC. fusca var.vacuolata (34°C) andC. kessleri (34–36°C) assuming an intermediate position. Thus, there is no wide gap in the temperature limits between the normal (“low-temperature”) species ofChlorella and the “high-temperature” species,C. sorokiniana.     

A comparative description of mitochondrial DNA differentiation in selected avian and other vertebrate genera

Levels of mitochondrial DNA (mtDNA) sequence divergence between species within each of several avian (Anas, Aythya, Dendroica, Melospiza, and Zonotrichia) and nonavian (Lepomis and Hyla) vertebrate genera were compared. An analysis of digestion profiles generated by 13-18 restriction endonucleases indicates little overlap in magnitude of mtDNA divergence for the avian versus nonavian taxa examined. In 55 interspecific comparisons among the avian congeners, the fraction of identical fragment lengths (F) ranged from 0.26 to 0.96 (F = 0.46), and, given certain assumptions, these translate into estimates of nucleotide sequence divergence (p) ranging from 0.007 to 0.088; in 46 comparisons among the fish and amphibian congeners, F values ranged from 0.00 to 0.36 (F = 0.09), yielding estimates of P greater than 0.070. The small mtDNA distances among avian congeners are associated with protein-electrophoretic distances (D values) less than approximately 0.2, while the mtDNA distances among assayed fish and amphibian congeners are associated with D values usually greater than 0.4. Since the conservative pattern of protein differentiation previously reported for many avian versus nonavian taxa now appears to be paralleled by a conservative pattern of mtDNA divergence, it seems increasingly likely that many avian species have shared more recent common ancestors than have their nonavian taxonomic counterparts. However, estimates of avian divergence times derived from mtDNA- and protein-calibrated clocks cannot readily be reconciled with some published dates based on limited fossil remains. If the earlier paleontological interpretations are valid, then protein and mtDNA evolution must be somewhat decelerated in birds. The empirical and conceptual issues raised by these findings are highly analogous to those in the long-standing debate about rates of molecular evolution and times of separation of ancestral hominids from African apes.