A HUMAN DISPERMIC CHIMAERA FIRST SUSPECTED FROM ANALYSIS OF THE BLOOD GROUP GENE-SPECIFIED GLYCOSYLTRANSFERASES

The red cells of a normal male blood donor, K.S., were first grouped as B but he was found to lack anti-A in his serum. Closer investigation revealed that his red cells had very weak A activity, demonstrable only by absorption and elution of anti-A. He is a non-secretor of ABH and a secretor of Le^a. Blood group A-, B and H-gene specified glycosyltransferases were detected in his serum. In contrast to the finding of a B antigen of normal strength on his red cells, the B transferase in his serum was only about 30% of the normal level and, despite the very weak A activity of K.S.'s red cells, the A transferase level was about 50% of that found in the serum of group A individuals with normal strength of A antigen. Moreover, the A transferase on the basis of its pH optimum, Km values for donor and acceptor substrates, activation by divalent cations, isoelectric focusing profile and capacity to convert O to A-active cells, was characterized as the product of an A¹ gene. A family study showed that K.S.'s wife is group A₂ and that they have two sons, one group A₂ and the other group B. The group B son is assumed to have inherited a B gene from the propositus but the level of B transferase in the son's serum is three times as high as that in his father's serum. The wife of the propositus and his group A₂ son have normal A² transferases in keeping with their A₂ red cell status. The A₂ son therefore appears to have inherited an A² gene from his mother but neither the A¹ nor the B gene shown to be carried by his father. The distribution of transferase activities in K.S.'s red cells differs from that in his serum. A level of B transferase within the normal range was found in his red cell membranes but a very low level of A transferase was detected. The discrepancies between the serum transferases and ABO-red cell group, together with the pattern of inheritance within the family, led to a suspicion of chimaerism. This was confirmed by the finding of fibroblasts with the female 46XX karyotype in cultures of the propositus' skin. These results suggest that K.S. is a dispermic chimaera with two different cell lines of the genotypes BO and A¹O or A¹A¹. The group A₂ son is assumed to have inherited an O gene from his father. It seems probable that K.S.'s bone marrow and reproductive organs are comprised predominantly of the XY cell line which carries the blood group BO genotype whereas his skin and other tissues which contribute the A¹ transferase to his plasma, are partly made up of the XX cell line which carries the blood group A¹O or A¹A¹ genotype.     

α-N-ACETYL-d-GALACTOSAMIYL- AND α-d-GALACTOSYLTRANSFERASE ACTIVITIES IN SERA OF CIS AB BLOOD GROUP INDIVIDUALS

Thirteen cis AB persons from five families were examined for serum glycosyltransferase activities associated with the biosynthesis of A and B blood group characters. Their transferases were generally homogeneous within one family, except for A₂/cis AB genotypes, whose A enzyme level was similar to the A₂ normal sera, but they varied from one family to another. These activities differed quantitatively and qualitatively from A, B and AB normal sera. Studies of A transferase showed variations in the pH-dependent curve, the effect of cofactors and the capacity of conversion of O red cells into A-active cells. Moreover, A and B transferases behaved differently with respect to their relative levels than did AB heterozygous normal sera. The results were discussed and it was suggested that a mutation of a single enzyme transferring both galactose and N-acetyl-galactosamine could explain these properties.     

Detection of A1A2 and A2AAm1 heterozygotes among human A blood group phenotypes.

From the variations of alpha-N-acetylgalactosaminyltransferases activities with the pH, evidence was obtained for the recognition of A1A2 heterozygotes in normal A blood group sera. Besides, unusual transferase properties associated with two A2 sera from individuals out of AAm1 siblings, lead to the identification of the very infrequent A2AAm1 genotypes. These results strongly support the simultaneous coexistence of both A1 and A2 transferases in heterozygotes' sera, and bring some new information on the genetical background of the Am phenotype. The meaning of transferase properties directly determined on whole sera is briefly discussed.     

STUDY OF THE α-N-ACETYLGALACTOSAMINYLTRANSFERASE IN SERA AND RED CELL MEMBRANES OF HUMAN A SUBGROUPS

The product of the A blood group gene in the erythrocyte membrane and serum from ‘weak A’ variants was investigated using low and high molecular weight acceptors and compared with common A₁ and A₂ blood samples. A reduced enzyme activity was detected, but only in some of the A variants, namely the A₃ (eight out of eleven), A_m or A_y samples. Other sera from A₃ (three out of eleven), A_x, A_end or A_el individuals were apparently devoid of enzyme activity. A kinetic study by temperature inactivation of A blood group sera also showed that A enzymes are more labile than B enzymes from B_I or B_II sera. Moreover, in one case (A₃ sample Del.), a very fast inactivation of the A enzyme was observed, suggesting the occurrence of a variant enzyme qualitatively different from the others so far studied. The erythrocyte membrane preparations from all A variants contained no detectable A enzyme activity except for the A₁ and A₂ samples, the former being three to five times more active than the latter. The B enzyme activities from four samples of B RBC tested were comparatively stronger than the A enzyme activity of A₁ RBC. The results were discussed and it was suggested that the synthesis and/or the secretion of the A enzyme in the organism is not uniform from one tissue to another, but could depend on which of the ‘weak A’ alleles or modifier genes is involved.     

Methylated 5'-terminal caps of adenovirus type 2 early mRNA: evidence for at least six 5'-termini.

The 5′-terminal caps of early adenovirus (Ad) 2 mRNA were isolated, fractionated, and compositionally analyzed. Cultured human KB cells were labeled with l-[methyl-³H]methi-onine and ³²Pi from 2 to 7 hr postinfection in the presence of cycloheximide. Labeled RNA was extracted from polysomes and fractionated into poly(A)⁺ and poly(A)^? molecules. Viral mRNA was isolated by hybridization to Ad2 DNA, then digested with RNase T2, and cap 1 [m⁷G(5′)ppp(5′)N^mpNp] and cap 2 [m⁷G(5′)ppp(5')N^mpN^mpNp] fractions were isolated by DEAE-Sephadex chromatography in 7 M urea. m⁷G was shown to be located at the 5′ termini of early Ad2 mRNA by periodate oxidation and β elimination. Cap 1 and cap 2 fractions were fractionated by electrophoresis on DEAE-cellulose paper. The cap 1 fraction separated into three major spots (a, b, and d), in order of increasing negative charge), and the cap 2 fraction into four major spots (a, b, c, and d). The methylated constituents of each spot were determined and used to deduce the number and composition of individual caps. The following results were obtained. Cap 1 structures: Spot a is m⁷GpppU^mpN1; b contains m⁷GpppU^mpN2 (N1 and N2 are different unmethylated nucleosides) and m⁷GpppA^mpN; c is m⁷GpppN^1mpN, m⁷Gpppm⁶A^mpN, and m⁷GpppG^mpN (N^1m is m₂⁶A^m or an unknown base-modified 2′-O-methyl nucleoside). Cap 2 structures: Spot a is m⁷Gppp(U^mp,N^1mp)N1; b contains at least two components, combinations of G^m, U^m, N^1m, and m⁶A^m; c contains at least two components, combinations of G^m, U^m, A^m, and m⁶A^m; d is at least two components containing combinations of C^m, A^m, and m⁶A^m. No significant differences in methylated constituents were detected in early mRNA repared from infected cells in the presence or absence of cycloheximide. Although we detect at least 13 caps, the minimum number of distinct 5′ termini is 6, because (i) N^1m could be m₂⁶A^m (2′-0-methyl N⁶,N⁶-dimethyl adenosine), (ii) m⁶A^m and A^m could be undermethylated precursors to m₂⁶A^m, and (iii) cap 1 structures could represent cap 2 structures without methylation of the penultimate nucleoside. Therefore there may be from 6 to 13 distinct Ad2 early mRNAs, which is interesting considering that only four general areas of the genome (each close to the 3′ end of each early gene block) appear to function as promoters [Berk, A. J., and Sharp, P. A. (1977). Cell12, 45–55; Evans, R. M., et al. (1977). Cell12, 733–739]. Analysis of KB cell mRNA resolved about 12 cap 1 and 10 cap 2 structures. All Ad2 mRNA cap spots resolved by electrophoresis corresponded in mobility and composition to major cap spots found in cell mRNA. Thus, the methylated caps of early mRNA are similar to those of the host cell. In contrast, most late Ad2 mRNA molecules have an identical cap sequence [Gelinas, R. E., and Robert, R. J. (1977). Cell11, 533–544].     

Methylated 5′-terminal sequences of vaccinia virus mRNA species made in Vivo at early and late times after infection

The 5′ terminals of vaccinia virus mRNAs synthesized in vivo contain additional sites of methylation not found in vaccinia virus mRNAs synthesized in vitro, suggesting the possibility of host cell modifications. mRNAs synthesized in vitro by enzymes present in purified vaccinia virions contain 7-methylguanosine (m⁶G) at their 5′ terminals in structures of the type m⁷G(5′)pppN₁^m-N₂ in which N₁^m is exclusively 2′-O-methyladenosine(A^m) or 2′-O-methylguanosine (G^m) and N₂ is not methylated. Two additional sites of methylation were found in the 5′ terminals of vaccinia virus-specific mRNAs synthesized in HeLa cells. The adenosine residue in the penultimate position is frequently doubly methylated to form N⁶,2′-O-dimethyladenosine (m⁶A^m) and N₂ is often methylated at the 2′ position to form structures of the type m⁷G(5′)pppN₁^m-N₂^m. Both of these modifications also occur in the mRNA of the uninfected host cell. In virus-specific mRNAs synthesized at early times after infection, N₁^m is either m⁶A^m,A^m, or G^m, and N₂^m is A^m, U^m, C^m, G^m, or m⁶A^m. Late vaccinia virus-specific RNA has a much higher proportion of m⁶A^m and A^m and correspondingly less G^m in the penultimate (N₁^m) position compared to early mRNA. In addition, late mRNA has relatively little methylation at the third (N₂) position of which a very high proportion is A_m. Both temporal classes of viral mRNA differ from the mRNAs of uninfected HeLa cells in lacking U^m or C^m in the penultimate position and having far more A^m in the third position. In early and late virus-specific mRNA species there were approximately one 5′ terminal per 1000 nucleotides and one 5′ terminal per 1250 nucleotides, respectively.     

Activation of adenosine A1 and A2 receptors differentially affects acetylcholine release from electric organ synaptosomes by modulating calcium channels

Adenosine inhibited the release of acetylcholine (ACh) evoked by high K⁺ depolarization from synaptosomes isolated from the electric organ of the Japanese electric ray Narke japonica. The adenosine A₁ receptor agonist N⁶-cyclohexyladenosine was an effective inhibitor. Conversely, in the presence of an A₁ receptor antagonist, 8-cyclopentyltheophylline, adenosine potentiated the release of ACh. The A₂ receptor agonist N⁶-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl] adenosine also facilitated the evoked ACh release. Thus, adenosine inhibits the evoked release of ACh via the A₁ receptor while it facilitates the release via the A₂ receptor. The EC₅₀ for inhibition and facilitation by adenosine was about 1 and 41 μM, respectively. There are three known types of calcium channels (N-, P/Q- and L-type) in synaptosomes. The effects of Ca²⁺ channel type-specific blockers on the modulation of ACh release by adenosine A₁ or A₂ receptor activation revealed that inhibition by A₁ receptor activation was caused via inhibition of N-type calcium channels and the facilitative effects by A₂ receptor activation was mediated by potentiation of P-type calcium channels.     

The Effects of Hypothyroidism on 5-HT1A and 5-HT2A Receptors and the Serotonin Transporter Protein in the Rat Brain

The effects of hypothyroidism on 5-HT_1A and 5-HT_2A receptors and the serotonin transporter protein were studied in thyroidectomized male Wistar rats in two experimental groups: 1) animals kept on an iodine-free diet (hypothyroid rats) and 2) animals kept on thyroxine (15 g/kg) for 21 days (giving normal thyroid hormone levels, euthyroid animals). Sham-operated rats served as controls. Binding of [³H]8-OH-DPAT with 5-HT_1A receptors and [³H]citalopram with the transporter protein in the hippocampus and midbrain showed no changes in hypothyroid rats as compared with controls. Conversely, there were significant decreases in [³H]ketanserin binding to 5-HT_2A receptors in the frontal cortex in hypothyroid rats as compared with controls; this decrease was reversed by thyroxine treatment. Thus, losses of cortical 5-HT_2A receptors appears to be the main consequence of hypothyroidism at the level of the serotonin system of the brain.     

Thalassemia and erythroid transcription factor KLF1 mutations associated with borderline hemoglobin A2 in the Thai population

IntroductionElevated hemoglobin (Hb) A₂ is an important diagnostic marker for β-thalassemia carriers. However, diagnosis of cases with borderline Hb A₂ may be problematic. We described the molecular characteristics found in a large cohort of Thai subjects with borderline Hb A₂.Material and methodsExamination was done on 21,657 Thai subjects investigated for thalassemia at Khon Kaen University, Thailand. A total of 202 subjects with borderline Hb A₂ (3.5–4.0%) were selectively recruited and hematological parameters were recorded. DNA variants in α-, β-, δ-globin, and Krüppel-like factor 1 (KLF1) genes were examined using PCR.ResultsAmong 202 subjects, DNA analysis identified carriers of α⁺-thalassemia (n = 48; 23.8%), β-thalassemia (n = 22; 10.9%) and KLF1 mutations (n = 48; 23.8%). No molecular defect was observed in the remaining 84 (41.5%) subjects. Interaction of KLF1 and α-thalassemia was observed in 10 cases. Of the 22 β-thalassemia carriers, five β⁺-thalassemia mutations were identified with lower MCV and higher Hb A₂. Seven KLF1 mutations were detected in 10 genotypes in subjects with higher MCV and Hb F. No β⁰-thalassemia, α-globin gene triplication or δ-globin gene mutation was detected.ConclusionsA large proportion of subjects with borderline Hb A₂ are not β-thalassemia carriers and for those with β-thalassemia, only mild β⁺-thalassemia mutations were detected. Evaluation of the patients using Hb A₂, Hb F and MCV values will help in selecting cases for further molecular analysis. The results should explain the unusual phenotype of the cases and facilitate a thalassemia screening program in the region.     

Effect of mercaptans and other agents on the human complement component β1C-globulin

Immunoelectrophoretic analyses and complement titrations of whole human serum show that a number of physical, chemical and immunological agents affect the β_1C/β_1A-globulin system which is considered to represent the third component of complement (C′₃). The transformation from β_1C-globulin to β_1A-globulin is the normal result of ageing, while antigen-antibody complexes, polylysine, hydrazine and cobra venom accelerate this change. In addition, α-globulin fragments of β_1A-globulin appear after interaction of normal sera with antigen-antibody complexes, treatment with cobra venom, and by storage under nitrogen in glass tubes or in polyethylene containers. Similar α-fragments are seen in the aged sera of some patients with glomerulonephritis or renal allografts. With prolonged storage these α-fragments can reform β_1A-globulin. On the other hand, 2-mercaptoethanol and penicillamine produce complete dissolution of β_1C-globulin into rapidly migrating, poorly defined fragments in the α-globulin and albumin regions, and transform β_1A-globulin into a stable β₂-globulin. The reduced fragments of β_1C-globulin, if not alkylated, can subsequently form the β₂-globulin, but not the β_1A-globulin. These results indicate that β_1C-globulin is composed of several subunits, some of which are joined by disulphide bonds, but that the α-globulin fragments seen in some pathological sera are not a result of disulphide bond reduction.     

Zinc (Zn2+) binds to and stimulates the activity of group i but not group II phospholipase A2

Phospholipase A₂ plays an important part in the generation of inflammatory lipid mediators and so it is of major interest to understand functional distinctions between structurally similar forms of phospholipase A₂. In the present study, the influence of zinc (Zn²⁺) on the activity of group I and group II phospholipase A₂ was examined in vitro. It appeared that Zn²⁺ (0.04–1×10^–3 M) increased group I phospholipase A₂ activity from porcine pancreas and rat lung whereas the activity of group II phospholipase A₂ fromCrotalus atrox andVipera russelli was unaffected. The presence of Cd²⁺ of Hg²⁺ (0.8–5×10^–3 M) also increased group I pancreatic phospholipase A₂ activity while no augmentation was found with Cr²⁺, Fe²⁺ or Mg²⁺. The selective stimulation of group I phospholipase A₂ by Zn²⁺ corresponded to a binding of these phospholipases A₂ to a zinc-affinity column, while group II phospholipase A₂ was not bound. Furthermore, the PLA₂ activity in bronchoalveolar lavage fluid from rat was stimulated by Zn²⁺. These results indicate that Zn²⁺ binds to and increases the activity of group I, but not group II phospholipase A₂. This difference in Zn²⁺-binding may be used to discriminate between group I and group II phospholipase A₂ and to separate the enzymes from each other in complex biological materials. The possibility that activation of group 1 phospholipase A₂ in the lung is important in zinc-induced metal fume fever is implied.     

Graph theory of configurational and viscoelastic properties of polymers, 2. Linear polymer chains containing small copolymer blocks

The graph theory of configurational and viscoelastic properties of polymers is extended to treat block copolymer chains. The analytical expressions for zero-shear-rate viscosity, η₀, and steady-state recoverable compliance, J_e⁰, two important linear viscoelastic properties of polymers, are obtained for B_mA₂B_m, AB_mA and B_lAB_m type block copolymer chains. When the structure of a linear polymer is altered locally by incorporating a small amount of another monomer, the variation of the viscoelasticity cannot be ignored, their variation being strongly dependent on the block structure. Thus, the results obtained may prove useful for understanding and interpreting rheological data of block copolymers.     

The cross-reacting antibodies of group O sera: immunological studies and possible explanation of the observed facts

Eluates were prepared from fifteen group O sera using A and B red cells. The sera originated from group O women who had either A or B children. In eight of the chosen sera, anti-A was the immune antibody and in seven, the anti-B. Antibody cross-reacting with both A and B red cells was found in all the eluates.

The results of titrations of cross-reacting antibody eluted from the red cells, both before and after absorption with A or B red cells and group specific substances, all indicate that the cross-reacting antibody produced by stimulation with antigen A is different from that produced by B.

The work also includes experiments using A₂, A_x and A_m red cells in conjunction with an immune group O serum chosen because of its good reactivity with A_x and A_m cells. It is particular interest that potent eluates from this serum prepared from A₁ cells failed to agglutinate cells of A_x slightly less potent eluates from group B agglutinated A_x to a titre of 128.

Two types of cross-reacting antibodies are postulated depending upon whether they are produced in response to stimulation by A or B. It is thought that most of the cross-reactive antibodies are directed against common portions of the blood group active oligosaccharide side chains, including the substituent at carbon atom 2 of the terminal non-reducing sugar (A (NH.CO.CH₃) or B(OH) specificity).

Denaturation or alteration of the spatial configuration of the antibody molecules after dissociation from the antigen may account for some of the observed phenomena.

     

Distribution patterns of apolipoproteins A1, A2, and B in the wall of atherosclerotic vessels

Summary Atherosclerotic vessels were analysed histochemically for distribution, quantity, and composition of apolipoprotein (Apo) types in the vascular wall. The specimens comprised all stages of atherosclerosis, from very discrete intimal changes to complicated lesions. The vessel specimens were marked with antibodies against human Apo A₁, A₂, and B. Apo A₁ can be demonstrated in even the earliest stage of atherosclerosis, and increases with the progression of the disease. In the initial stage, Apo A₁ is found first in lumen-adjacent layers of the intima, and is evident in deeper layers of the wall as the disease progresses. Arteries of muscular type show accumulation of Apo in an earlier stage (or in greater quantity at the same stage) than arteries of elastic type. At all stages, the amount of Apo A₁ always exceeds that of A₂ and B. In the intima, Apo B is higher than Apo A₂, the media contains hardly any Apo B, and the adventitia has less B than A₂. Within the intimal layer, Apo A₁ and A₂ are found in an intracellular (mainly in foam cells) or in an extracellular location, according to the stage of atherosclerosis. Apo B is almost exclusively extracellular; only cases of advanced atherosclerosis show some intracellular localization (mostly in foam cells), visualized as electron dense lamellar organelles, probably of lysosomal origin. In the media, Apo A₁ and A₂ are accumulated in intracellular deposits, whereas the extracellular storage of Apo A₁ A₂ and B is observed only in cases with the most severe damage. Our investigations suggest that the accumulation of apolipoproteins in the vascular wall is effected not only by insudation from the plasma, but also by neosynthesis and/or metabolism by locally derived cells or cells immigrating in the process of atherosclerosis. The presence of Apo A₁ and A₂ in the vessel wall is now documented, and their role at this site apparently differs from that in the plasma.Dedicated to Professor E. Grundmann on the occasion of his 70th birthday.     

Identification ofEntamoeba histolytica intracellular phospholipase A and lysophospholipase L1 activities

Entamoeba histolytica phospholipase A and lysophospholipase activities from a vesicular subcellular fraction (P30) were analyzed. The products, obtained using specific substrates labeled with¹⁴C or³H, indicated the presence of phospholipase A₁ and A₂ as well as lysophospholipase L₁ activities. The enzymes detected could participate in phospholipid metabolism and the alkaline phospholipase A₂ may contribute toE. histolytica cytopathogenicity.     

Time-resolved fluoroimmunoassay of pancreatic phospholipase A2

Summary We have developed an immunoassay for human pancreatic secretory phospholipase A₂ based on time-resolved fluorescence. The labeled antibody technique in combination with the time-resolved fluorometric detection of the Europium label, which essentially eliminates all background fluorescence, resulted in a high sensitivity and a wide linear range. Our assay is a one-incubation, multi-site, solid-phase assay on polystyrene micro-titer strips, even though a polyclonal antibody was used. Increased levels of immunoreactive phospholipase A₂ are found by this assay in sera of patients suffering from acute pancreatitis.     

Two different acto-S1 complexes

Summary Based on change in anisotropy of fluorescently labelled S1 and on increase in turbidity of acto-S1 complex when S1 bound to F-actin, we reported previously that depending on the molar ratio of S1 to actin two different complexes of actin monomer (A) and myosin subfragment 1 (S1) could be formed: A₁ ^*S1 (one actin with one S1) and A₂ ^*S1 (two actins with one S1). Here we extend these findings to F-actin labelled with pyrene and cross-linked to S1 with 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC). The fluorescence of pyrene F-actin decreased with increase in S1 concentration and reached saturation at a molar ratio of S1 to actin of either 0.5 or 1.0, depending on whether S1 was added slowly (5 min) or quickly (10–20 s between additions). Incubation of A₂ ^*S1 complex in excess of S1 for > 1 h caused a shift in equilibrium towards the A₁ ^*S1 complex. The A₂ ^*S1 complexes were not formed at high S1 to actin ratios (>1.0) owing to competition between heads. Crosslinking experiments showed that the formation of EDC crosslinked products, 175–185 kDa doublet and 265 kDa band, depended on the ratio S1 to actin. To assess the relative ratio of S1 and actin in crosslinked products, we labelled S1 and F-actin with different fluorescent probes (5-IAF and IATR). The S1 to actin ratio was proportional to the ratio of intensities of fluorescence of labelled S1 and actin. The S1 to actin ratio in 265 kDa product was two times smaller than in 175–185 kDa doublet (which is believed to be A₁ ^*S1 complex) and therefore 265 kDa band corresponded to A₂ ^*S1. Transition between two types of binding may be important to understanding how muscle contracts.     

A spin-probing study of non-magnetic ionomers: Cu2+ -containing polyethylene/methacrylic acid copolymer neutralized with Zn2+

A spin-probing technique was first applied to a study of a non-magnetic ionomer, polyethylene/methacrylic acid copolymer neutralized with Zn²⁺ (E-MAA-Zn); 2 mol-% Cu²⁺ ions were incorporated in replacement of Zn²⁺, and its ESR spectra were observed in detail at various temperatures in order to obtain physico-chemical properties of E-MAA-Zn, especially its structural changes. We found no significant influence of the 2 mol-% Cu²⁺ -incorporation upon the properties of the Zn²⁺ ionomer by differential scanning calorimetry (DSC), which showed two endothermic peaks, (1) an irreversible one at lower temperature which is associated with the ionic aggregate phase in the ionomer, (2) a reversible one at higher temperature with considerable thermal hysteresis which is due to melting of the polymer main chains. ESR hyperfine coupling constants (A_∥ and A_⊥) of Cu²⁺ showed no sign of structural changes of the polymer around the phase-transition temperatures, whereas ESR g-values (g_∥ and g_⊥) of Cu²⁺ proved the onset of the softening of the polymers to be attributable to polymer melting. The latter slope-changes in the temperature dependences were reversible, although with considerable thermal hysteresis. These findings are compatible with the DSC data. As for the irreversibility regarding the endotherm at lower temperature, broad ESR hyperfine components recovered gradually, indicating an order-disorder nature of the ionic aggregate phase transition.     

Changes in 5-HT2A-mediated behavior and 5-HT2A- and 5-HT1A receptor binding and expression in conditional brain-derived neurotrophic factor knock-out mice

Changes in brain-derived neurotrophic factor (BDNF) expression have been implicated in the etiology of psychiatric disorders. To investigate pathological mechanisms elicited by perturbed BDNF signaling, we examined mutant mice with central depletion of BDNF (BDNF^2L/2LCk-cre). A severe impairment specific for the serotonin 2A receptor (5-HT_2AR) in prefrontal cortex was described previously in these mice. This is of much interest, as 5-HT_2ARs have been linked to neuropsychiatric disorders and anxiety-related behavior. Here we further characterized the serotonin receptor alterations triggered by BDNF depletion. 5-HT_2A ([³H]-MDL100907) and 5-HT_1A ([³H]-WAY100635) receptor autoradiography revealed site-specific alterations in BDNF mutant mice. They exhibited lower 5-HT_2A receptor binding in frontal cortex but increased binding in hippocampus. Additionally, 5-HT_1A receptor binding was decreased in hippocampus of BDNF mutants, but unchanged in frontal cortex. Molecular analysis indicated corresponding changes in 5-HT_2A and 5-HT_1A mRNA expression but normal 5-HT_2C content in these brain regions in BDNF^2L/2LCk-cre mice. We investigated whether the reduction in frontal 5-HT_2AR binding was reflected in reduced functional output in two 5-HT_2A-receptor mediated behavioral tests, the head-twitch response (HTR) and the ear-scratch response (ESR). BDNF^2L/2LCk-cre mutants treated with the 5-HT_2A receptor agonist (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) showed a clearly diminished ESR but no differences in HTR compared to wildtypes. These findings illustrate the context-dependent effects of deficient BDNF signaling on the 5-HT receptor system and 5-HT_2A-receptor functional output.     

Deletion of the adenosine A(2A) receptor in mice enhances spinal cord neurochemical responses to an inflammatory nociceptive stimulus

Knockout mice lacking the adenosine A_2A receptor are less sensitive to nociceptive stimuli, and this may be due to the presence of pronociceptive A_2A receptors on sensory nerves. In support of this hypothesis, we have recently shown that in A_2A receptor knockout mice there are marked reductions in the changes of two markers of spinal cord neuronal activity, [³H]MK801 binding to NMDA receptors and uptake of [¹⁴C]-2-deoxyglucose, in response to formalin injection. We now report that following a more prolonged inflammatory stimulus, consisting of intraplantar injections of PGE₂ and paw pressure, there was in contrast an increase in [³H]MK801 binding and [¹⁴C]-2-deoxyglucose uptake in the spinal cords of the A_2A receptor knockout mice which was much greater than in the wild-type mice. This increase suggests that when there is a pronounced inflammatory component to the stimulus, loss of inhibitory A_2A receptors on inflammatory cells outweighs the loss of pronociceptive A_2A receptors on peripheral nerves so that overall there is an increase in nociceptive signalling. This implies that although A_2A antagonists have antinociceptive effects they may have only limited use as analgesics in chronic inflammatory pain.