Characterisation of the Glycine Modulatory Site of the N-Methyl-d-Aspartate Receptor-Ionophore Complex in Human Brain

[3H]Glycine binding and glycine modulation of [3H]MK-801 binding have been used to study the glycine allosteric site associated with the N-methyl-D-aspartate receptor complex in postmortem human brain. The effect of glycine on [3H]MK-801 binding appeared sensitive to duration of terminal coma, and possibly postmortem delay. Thirty percent of the binding occurred in a subfraction of brain tissue and did not show enhancement by glycine and glutamic acid. [3H]Glycine binding to a subfraction free from this component was studied and showed high specific binding. KD and Bmax values showed considerable intersubject variability which did not appear to be due to demographic features or to tissue content of amino acids with an affinity for this site. The pharmacological characteristics of binding in this subfraction and a correlation between Bmax values and the maximal enhancement of [3H]MK-801 binding by glycine are consistent with [3H]glycine binding occurring to an N-methyl-D-aspartate receptor complex associated site. Further support for this is provided by a significantly lower Bmax value for [3H]glycine binding in subjects with Alzheimer's disease and reduced glycine enhancement of [3H]MK-801 binding. However, the effect of perimortem factors makes it difficult to confidently attribute this solely to a disease-related change in the receptor. The possible role of the glycine allosteric site in the treatment of neuropsychiatric disorders is discussed.     

Inhibition of phosphoinositide turnover by praziquantel in Schistosoma mansoni.

The effect of praziquantel on phosphoinositide turnover was examined in Schistosoma mansoni to determine if this anthelminthic modulates signal transduction pathways in parasites. Adult worms were radiolabeled with [3H]myoinositol for 24 hr and total inositol phosphate levels determined in the presence of praziquantel. Praziquantel inhibited inositol phosphate turnover when activated with NaF plus AlCl3 or with the nonhydrolyzable guanine nucleotide-binding protein analogue GTP gamma S. Furthermore, praziquantel decreased basal turnover of inositol phosphates. Inhibition was seen in both male and female worms as well as in schistosomula. These data indicate that inhibition of phosphoinositide turnover may contribute to the effect of praziquantel on parasite survival within the definitive host.     

Visualization and solubilization of rat brain opiate receptors with a “κ” ligand selectivity pattern

1. Specific binding of [3H]ethylketocyclazocine (EkappaC), a prototype kappa-opiate agonist, to slide-mounted rat striatal sections is increased in the presence of 100 mM NaCl at 4 degrees C. 2. Under similar incubation conditions, binding of mu and delta prototype opiates is reduced to almost undetectable levels. 3. Correlation (P less than 0.01) of the ligand selectivity pattern of [3H]EKC displacement with the potencies of various opiate drugs in inhibiting the contractions of the rabbit vas deferens, a kappa-opiate receptor bioassay, suggests that the binding site under study represents the pharmacologically relevant kappa-opiate receptor. 4. Visualization of these kappa-opiate receptors with tritium-sensitive film reveals a striking, highly discrete brain distribution pattern (e.g., striatal patches, habenular stripe) which is similar to that of [3H]dihydromorphine and [3H]naloxone. 5. Soluble [3H]EKC binding sites obtained from rat membranes also possess a kappa-like ligand selectivity pattern, with bremazocine being a potent displacer while mu and delta ligands are almost inactive. 6. A possible explanation of these data is that the "kappa"-opiate binding site in rat brain is one transitional state of an opiate receptor capable of assuming distinct conformations with characteristic ligand selectivity patterns. Other possibilities such as pre and post-synaptic locations should also be considered.     

Carbon dioxide exchange characteristics of C4 Hawaiian Euphorbia species native to diverse habitats

Summary The characteristics of the photosynthetic apparatus of 11 Hawaiian Euphorbia species, all of which possess C₄ photosynthesis but range from arid habitat, drought-deciduous shrubs to mesic or wet forest evergreen trees and shrubs, were investigated under uniform greenhouse conditions. Nine species exhibited CO₂ response curves typical of C₄ plants, but differed markedly in photosynthetic capacity. Light-saturated CO₂ uptake rates ranged from 48 to 52 mol m^-2 s^-1 in arid habitat species to 18 to 20 mol m^-2 s^-1 in mesic and wet forest species. Two possessed unusual CO₂ response curves in which photosynthesis was not saturated above intercellular CO₂ pressures [p(CO₂)] of 10 to 15 Pa, as typically occurs in C₄ plants.Both leaf (g₁) and mesophyll (g_m) conductances to CO₂ varied widely between species. At an atmospheric p(CO₂) of 32 Pa, g₁ regulated intercellular p(CO₂) at 12–15 Pa in most species, which supported nearly maximum CO₂ uptake rates, but did not result in excessive transpiration. Intercellular p(CO₂) was higher in the two species with unusual CO₂ response curves. This was especially apparent in E. remyi, which is native to a bog habitat. The regulation of g₁ and intercellular p(CO₂) yielded high photosynthetic water use efficiencies (P/E) in the species with typical CO₂ response curves, whereas P/E was much lower in E. remyi.Photosynthetic capacity was closely related to leaf nitrogen content, whereas correlations with leaf morphological characteristics and leaf cell surface area were not significant. Thus, differences in photosynthetic capacity may be determined primarily by investment in the biochemical components of the photosynthetic apparatus rather than by differences in diffusion limitations. The lower photosynthetic capacities in the wet habitat species may reflect the lower light availability. However, other factors, such as reduced nutrient availability, may also be important.     

Replication of bacteriophage lambda DNA. Examination of variants containing double origins and observation of a bias in directionality

Bacteriophage λ variants have been constructed that possess two λ ori sites. Replicative intermediates resulting from infection with these phages have been investigated. We find that initiation of replication from the ori site on an EcoRI fragment (containing all the DNA sequences from within the red gene to the middle of gene O) cloned in the inverted orientation is predominantly bidirectional but occurs at a decreased frequency. Double initiations were observed at low frequency. However, a second cloned ori fragment (carrying two large deletions and a small insertion) cloned in the normal orientation demonstrated insignificant levels of replication from the cloned site unless the normal ori had already initiated.A bias in directionality of λ replication has been observed. Molecules that replicate unidirectionally propagate to the right more often than to the left. If the cloned ori-containing EcoRI fragment is inserted with reversed polarity, then the bias is towards the left. Bidirectional λ replicative intermediates also appear to show a similar bias but this is superimposed on a large, apparently random, effect that results in asymmetric growing-point propagation.     

Structure of Sendai viral proteins in plasma membranes of virus-infected cells.

Purified plasma membranes attached to polycationic polyacrylamide beads by their external surface were isolated from BHK cells infected with Sendai virus. Each of the viral proteins could be identified in the membranes of infected cells. Proteolysis with trypsin, which digests only the cytoplasmic surface of these membranes (because the external surface is protected by its attachment to beads), revealed that the internal proteins, L, P, NP, and M, were present on the cytoplasmic surface of the membrane and that small segments of the viral envelope glycoproteins, HN and F0, were partially exposed on the cytoplasmic surface. Since the major portions of HN and F0 are known to be present on the external membrane surface, these glycoproteins are transmembrane proteins before Sendai virus budding in infected cells.     

Rabbit heavy chain haplotypes — Allotypic determinants expressed byVH-CH recombinants

This report summarizes our current understanding of the heavy chain haplotypes found in our laboratories' rabbits. Independently derived data from several laboratories have been synthesized into a consistent picture of the linked inheritance of allotypic markers found on the different heavy chain classes and subclasses of rabbit immunoglobulins in pedigreed rabbits, including the families of three apparentVH-CH recombinants. In one recombinant, the entire group ofCH markers (C, C, and C) recombined with the set ofVH. Although in the other two recombinants all CH markers may also have recombined as a group, in one of these only IgG and IgACH genes were informative; in the other recombinant, only the IgG allotypes were informative. Some allotypic determinants found on IgM molecules (conformational) appear only when a specific variable region allotype (VHa) is combined with a specific constant region allotype (C). New combinations ofVHa and C allotypes were generated in two of the genetic recombinants and led to new conformational determinants. The gains and losses observed lend support to the hypothesis that the determinants result from conformations generated by the combination of allotype-specificVH and C protein sequences. Conceivably, DNA events that joinVH to diversity (D)- and joining (J)-coding sequences or mRNA processing events that splice J to C could be involved in generating the sequences that form allotype-specific determinants.     

Effects of butylated hydroxyanisole on the aryl hydrocarbon hydroxylase of rats and mice

The effects of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on the aryl hydrocarbon hydroxylase (AHH) activities in the liver, lung and skin of rats and mice have been studied to examine the possible mechanisms of the anticarcinogenic actions of these compounds. Both compounds inhibit the hydroxylase activities of hepatic microsomes and nuclei, with BHA a more potent inhibitor than BHT. The AHH of lung microsomes is inhibited to a lesser extent by BHA and BHT than that of the liver. The AHH activities of both liver and lung microsomes become less susceptible to the inhibition after pretreatment of the animals with 3-methylcholanthrene (MC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) but phenobarbital (PB) pretreatment does not produce such an effect. In skin homogenates, however, the AHH activities of control rats and mice are not inhibited by BHA and BHT. The only skin sample which is inhibited by BHA and BHT is that from TCDD-pretreated mice. It has been established that the extent of inhibition with different samples is related to the concentration of BHA in the incubation but not to the amounts or specific activities of microsomes used. Double reciprocal plots suggest that BHA exerts a mixed inhibition on the hydroxylase of liver microsomes with a K_i of 7.7 μM. Analysis of the metabolites of benzo[a]pyrene (BP) shows that BHA inhibits the formation of various metabolites uniformly without changing the regio-selectivity of the enzyme system. The mechanism of inhibition has also been studied with a reconstituted AHH system consisting of cytochrome P-450 (P-450), reductase and phospholipid. The system with P-450 isolated from PB-induced microsomes is inhibited to a much greater extent than that with MC-induced P-450. The results indicate that the inhibitory action of BHA is dependent on the species of the animal, tissue types and treatment with inducers.     

Specificity of Insertion by the Translocatable Tetracycline-Resistance Element Tn10

Genetic analysis of 131 independent transpositions of the tetracycline-resistance element Tn10 from a single site in phage P22 into the histidine operon of Salmonella typhimurium reveals that Tn10 insertions are not randomly distributed along this chromosomal target. The insertions occur in 22 different "clusters"; insertions within each cluster are very tightly linked in recombination tests. Tn10 insertions are not evenly distributed among the identified clusters. The existence of these clusters suggests that this chromosomal target contains particular genetic signals that guide Tn10 to particular preferred positions for insertion. Insertions within each cluster occur in both orientations with roughly equal frequency.--The relationship among different insertions within each cluster has been examined. The resolution of genetic mapping places an upper limit of about 50 basepairs on the distance between different insertions within a cluster. Different insertions within a cluster usually have the same reversion frequency; however, heterogeneity in reversion frequency has been detected in at least two clusters. For most clusters, the available data are consistent with the simple possibility that all insertions within a cluster are at identical positions; however, the data do not exclude other possibilities.