[3H]MK-801 Labels a Site on the N-Methyl-D-Aspartate Receptor Channel Complex in Rat Brain Membranes

The potent noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist [3H]MK-801 bound with nanomolar affinity to rat brain membranes in a reversible, saturable, and stereospecific manner. The affinity of [3H]MK-801 was considerably higher in 5 mM Tris-HCl (pH 7.4) than in previous studies using Krebs-Henseleit buffer. [3H]MK-801 labels a homogeneous population of sites in rat cerebral cortical membranes with KD of 6.3 nM and Bmax of 2.37 pmol/mg of protein. This binding was unevenly distributed among brain regions, with hippocampus greater than cortex greater than olfactory bulb = striatum greater than medulla-pons, and the cerebellum failing to show significant binding. Detailed pharmacological characterization indicated [3H]MK-801 binding to a site which was competitively and potently inhibited by known noncompetitive NMDA receptor antagonists, such as phencyclidine, thienylcyclohexylpiperidine (TCP), ketamine, N-allylnormetazocine (SKF 10,047), cyclazocine, and etoxadrol, a specificity similar to sites labelled by [3H]TCP. These sites were distinct from the high-affinity sites labelled by the sigma receptor ligand (+)-[3H]SKF 10,047. [3H]MK-801 binding was allosterically modulated by the endogenous NMDA receptor antagonist Mg2+ and by other active divalent cations. These data suggest that [3H]MK-801 labels a high-affinity site on the NMDA receptor channel complex, distinct from the NMDA recognition site, which is responsible for the blocking action of MK-801 and other noncompetitive NMDA receptor antagonists.     

Molecular organisation of the quinic acid utilization (QUT) gene cluster in Aspergillus nidulans

Summary The functional integrity of the QUTB gene (encoding quinate dehydrogenase) has been confirmed by transformation of a qutB mutant strain. The DNA sequence of the contiguous genes QUTD (quinate permease), QUTB and QUTG (function unknown) has been determined and analysed, together with that of QUTE (catabolic 3-dehydroquinase). The QUTB sequence shows significant homology with the shikimate dehydrogenase function of the complex AROM locus of Aspergillus nidulans, and with the QA-3 quinate dehydrogenase and QA-1S (repressor) genes of Neurospora crassa. The QUTD gene shows strong homology with the N. crassa QA-Y gene and QUTG with the QA-X gene. QUTD, QUTB, and QUTG, QUTE form two pairs of divergently transcribed genes, and conserved sequence motifs identified in the two common 5 non-coding regions show significant homology with UAS _GAL and UAS _QA sequences of the Saccharomyces cerevisiae and N. crassa Gal and QA systems. In addition, conserved 5 sequences homologous to the mammalian CAAT box are noted and a previously unreported conserved 22 nucleotide motif is presented.     

Rabbit MHC. II. Sequence analysis of the R-DP alpha- and beta-genes

Molecular genetics studies have recently revealed complexity in the MHC class II region that has not been detected by previous serologic and genetic studies. In humans, three subregions, DP, DQ, and DR, of the class II genes as well as the DZ alpha and DO beta genes, have been extensively characterized. Although homologs of these human genes were identified in many species, their expressibility has not been well defined in species other than the mouse. We have previously cloned the rabbit homologs of the HLA-DP alpha and beta genes whose protein products had never been detected. The sequences of rabbit DP alpha 1 and DP beta genes are reported herein and they indicate that the rabbit DP genes encode functional alpha- and beta-chains. Unfavorable nucleotides surrounding the first AUG codon may, however, reduce the translational efficiency of the R-DP beta mRNA and explain the difficulty in generating serologic reagents specific for rabbit DP molecules. A complex mutation in the beta 1 domain of the R-DP beta gene was similar to the one found in the H-2A beta 1 gene of five strains of mice. The origin of this mutation is discussed.     

Spectral analysis of ventilation in elderly subjects awake and asleep

We studied the periodicities of ventilation in elderly subjects using digital comb filtering. Two groups of subjects were studied, those with and without sleep apnea. Measurements were made in wakefulness, stage 1-2 sleep, and where possible in stage 3-4 sleep. For each of the digital filters we calculated the average power of the oscillatory output. To compare subject groups we first specifically determined the average power in the filter with the maximum output. The mean of this measurement was greater in elderly subjects with apnea compared with those without apnea, both during wakefulness and stage 1-2 sleep. In both groups of subjects the cycle time of the major ventilatory oscillations was on the order of 40-60 s. There was no difference in this cycle time between the two groups of subjects in wakefulness or stage 1-2 sleep. Thus, whereas similar oscillatory processes occur in subjects with and without apnea, it is the magnitude of the oscillation that differs between the two groups. These conclusions are supported by analysis of the output of individual filters of the digital comb filter. In both groups, stage 1-2 sleep produced significantly increased oscillations in ventilation. Both in wakefulness and stage 1-2 sleep, significantly greater periodicities occurred in the apneic compared with the nonapneic group. In the few subjects who had sufficient data in stage 3-4 sleep for spectral analysis, ventilatory oscillations were virtually absent in this state. Our data suggest that subjects who develop apnea during sleep have an increased propensity for periodic breathing even while awake.     

Enzyme-linked immunosorbent assays for the measurement of blood group A and B glycosyltransferase activities

ELISA assays have been developed for (1–3)N-acetylgalactosaminyltransferase (blood group A transferase) and (1–3)galactosyltransferase (blood group B transferase) activities. In these assays, microtitre plates coated with the bovine serum albumin conjugate of a synthetic Fuc1–2Gal-R acceptor substrate are incubated with the appropriate nucleotide donor (UDP-GalNAc or UDP-Gal) and human serum as the enzyme source. The resulting trisaccharide products Fuc1–2(GalNAc1–3)Gal-R-BSA or Fuc1–2(Gal1–3)Gal-R-BSA are detected and quantified with monoclonal antibodies selected not to cross-react with the substrate structure. With less than a microliter of human serum, product formation is proportional to enzyme concentration and to time of incubation of up to 90 min.     

Genetic analysis of a mutant class of Physcomitrella patens in which the polarity of gravitropism is reversed.

Summary In the moss Physcomitrella patens, single-cell protonemata and multicellular gametophores respond to reorientation relative to the gravity vector by growing negatively gravitropically. A mutant class in which the protonemata, but not the gametophores, respond by growing towards gravity has been identified. In this paper, we describe the isolation of additional mutants of this class. Complementation and segregation ratio analyses were carried out on these mutants, which indicate that a single gene may mutate to switch the polarity of gravitropism.     

Stable transformation of the moss Physcomitrella patens

Summary We report the stable transformation of Physcomitrella patens to either G418 or hygromycin B resistance following polyethylene glycol-mediated direct DNA uptake by protoplasts. The method described in this paper was used successfully in independent experiments carried out in our two laboratories. Transformation was assessed by the following criteria: selection of antibiotic-resistant plants, mitotic and meiotic stability of phenotypes after removal of selective pressure and stable transmission of the character to the offspring; Southern hybridisation analysis of genomic DNA to show integration of the plasmid DNA; segregation of the resistance gene following crosses with antibiotic-sensitive strains; and finally Southern hybridisation analysis of both resistant and sensitive progeny. In addition to stable transformants, a heterogeneous class of unstable transformants was obtained.     

Oral vaccination of skunks with raccoon poxvirus recombinants expressing the rabies glycoprotein or the nucleoprotein.

Twenty nine skunks (Mephitis mephitis) were vaccinated orally with raccoon poxvirus (RCN) recombinants: 10 with a recombinant expressing the rabies virus glycoprotein (RCNRG), 10 with RCNRG mixed with a recombinant expressing the rabies virus nucleoprotein (RCNRN) and nine with RCN alone. Rabies virus neutralizing antibodies were detected in six of the 20 skunks; five skunks (three given RCNRG, two given a mixture of recombinants) survived a rabies challenge that was lethal for nine skunks vaccinated with RCN alone.