A review of the in vitro and in vivo neurochemical characterization of the NMDA/PCP/Glycine/Ion channel receptor macrocomplex

Conclusions Current neurochemical studies of the NMDA receptor macromolecular complex are yielding new insights into the interactions of the subunits of this complex and the associated potential clinical benefits of selective modulation of these subnits. Such studies offer the great potential for a new generation of pharmacotherapies for a wide range of CNS disorders, including stroke, a condition for which there is currently no effective pharmacological treatment. However, it is essential to understand that the first generation products in this area may not be optimal pharmacotherapies, such that haracterization of possible receptor subtypes and understanding the molecular biology of the component proteins of the receptor complex will be crucial in the design of the optimal pharmacological modulators of the NMDA receptor complex.Special issue dedicated to Dr. Erminio Costa     

Post-castration rebound of an androgen regulated prostatic gene

Summary After castration, the rat dorsolateral prostate M-40 mRNA initially decreased then rebounded to precastrated levels. The cellular site of M-40 expression and its renewed expression after castration was defined by in situ hybridization histochemistry. In situ hybridization with either a ³²P-labeled or biotin-labeled M-40 cDNA probe demonstrated that M-40 mRNA levels were higher in the lateral than dorsal prostate. A second androgen regulated gene, RWB, also was highly expressed in the lateral prostate. The biotinylated cDNA probes provided microscopic resolution of the expressing cells, revealing two distinct morphologies of lateral epithelium which expressed both the M-40 and RWB mRNA. These morphologies appeared in ducts which contained either epithelial cell sheets that were highly convoluted or thinner epithelial cells with a minimal degree of convolution. The RWB mRNA decreased in both cell populations in response to androgen withdrawal. The decline and reappearance of M-40 mRNA also appeared in both epithelial cell types. These data demonstrated that after castration the M-40 mRNA initially decreased as expected for an androgen sensitive gene and then progressed to a fully inducible state. The mechanism of this progression remains to be elucidated.     

Purification of an antitrypanosomal factor fromPseudomonas fluorescens by high-pressure liquid chromatography

Studies on the purification of an antitrypanosomal factor (ATF-II) obtained fromPseudomonas fluorescens disclosed that high-pressure liquid chromatography (HPLC) with a Rad-pak porasil (10 silica) column and a GPC-60 Å column was an efficient procedure for the separation of the active components. Extraction of the factor with absolute ethanol prior to elution significantly enhanced the lytic activity of the HPLC eluates, as shown by marked pathologic changes followed by lysis in bioassays performed withTrypanosoma equiperdum. HPLC provided an increase of purification 30 times that obtained with gel filtration of the crude bacterial product. The lipopolysaccharide content of the purified fractions was markedly reduced and indicated an additional advantage for further in vivo tests in experimental infections withT. cruzi.     

Differential sensitivity to natural cell-mediated cytotoxicity of two rat colon adenocarcinoma variants differing in their tumorigenicity: identification of the effector cells as natural killer cells

Summary DHD/K12 TRb (PROb) and DHD/K12 TSb (REGb) are two cancer cell variants originating from the same rat colon adenocarcinoma. They differ in their tumorigenicity: when inoculated into syngeneic BDIX rats, PROb cells induce progressive tumors whereas REGb cells induce tumors which always regress. As previously described, there is an inverse relation between their tumorigenicity and their susceptibility to NCMC mediated by syngeneic spleen or peripheral blood lymphocytes: PROb cells are significantly less sensitive to NCMC than REGb cells. This suggests a role for NCMC in the regression of REGb tumors. In this work the BDIX NCMC effector cells active in vitro against REGb cells were identified as NK cells according to four criteria: (1) efficacy in a 4-h ⁵¹Cr release assay, (2) sensitivity to anti-asGM1 antibody plus complement, (3) LGL morphology, and (4) ability to bind with the same affinity REGb and YAC-1 cells. In spleen, these NK cells were heterogeneous with respect to their asGM1 surface density and their morphology. PROb cells were not lysed by these NK cells in a short-term cytotoxicity assay, but only in a 16-h assay. It was shown that PROb and REGb cells were bound with the same affinity by NK cells, thus they certainly differ in their ability to resist to NK lytic mechanisms. This difference could play a role in the different tumorigenicity of the two variants. Abbreviations used: NK, natural killer; NC, natural cytotoxic; NCMC, natural cell-mediated cytotoxicity; asGM1, asialo GM1; LL, large lymphocytes; LGL, large grnular lymphocytes; LAL, large agranular lymphocytes; PBMNC, peripheral blood mononuclear cells; E:T, effector to target cell ratio; C:H, cold to hot cell ratio; FBS, fetal bovine serum     

Long-delay learning in rats with parabrachial pontine lesions

Two experiments were conducted to test the hypothesis that ratswith lesions in the parabrachial nucleus of the pons (PbN) canacquire conditioned taste aversions only if the conditionedand unconditioned stimuli are presented close together in time.In experiment 1, rats with lesions in the medial PbN, lateralPbN or outside of the PbN (lesioned control), and unoperatedcontrol rats were trained to avoid Na-saccharin in a one-bottletest. In this procedure, a 5-min delay was imposed between the15-min Na-saccharin presentation and an injection of LiCl (0.3M, 1 % body weight, i.p.). Results showed that, after threeNa-saccharin-LiCl pairings, all rats, except the medial PbNgroup, acquired a strong aversion to Na-saccharin. In experiment2, the same rats were presented with LiCl (0.12 M) in their15-min daily access to fluid on 3 alternate days. Although ratsin the medial PbN group drank more LiCl on day 1 than rats inthe other groups, they significantly reduced their LiCl consumptionon day 2 and did not differ from other groups by day 3. Resultsare discussed in terms of possible behavioral and physiologicalmechanisms that might account for these phenomena.     

An experiment in enzyme evolution studies withPseudomonas aeruginosa amidase

The regulation of amidase synthesis inP. aeruginosa is under positive control. This review describes the experimental evolution of amidase and its regulator protein for the hydrolysis of novel substrates and experiments to elucidate the mechanism of the control system.     

Gravitropism in Higher Plant Shoots : V. Changing Sensitivity to Auxin

An alternative to the Cholodny-Went, auxin-transport hypothesis of gravitropic stem bending was proposed as early as 1958, suggesting that gravistimulation induces changes in sensitivity to auxin, accounting for differential growth and bending. To test the sensitivity hypothesis, we immersed marked, decapitated sunflower (Helianthus annuus L.) hypocotyl sections in buffered auxin solutions over a wide concentration range (0, 10⁻⁸ to 10⁻² molar IAA), photographed them at half-hour intervals, analyzed the negatives with a digitizer/computer, and evaluated surface-length changes in terms of Michaelis-Menten enzyme kinetics. Bending decreases with increasing auxin concentration; above about 10⁻⁴ molar IAA the hypocotyls bend down; increasing auxin inhibits elongation growth of lower surfaces (which is high at zero or relatively low auxin levels) but promotes upper-surface growth (which is low at low auxin levels). Thus, lower surfaces have a greater K_m sensitivity to applied auxin than upper surfaces. At optimum auxin levels (maximum growth), growth of bottom surfaces exceeds that of top surfaces, so bottom tissues have a greater V_max sensitivity. V_max sensitivity of vertical controls is slightly lower than it is for either horizontal surface; K_m sensitivity is intermediate. Clearly, gravistimulation leads to significant changes in tissue sensitivity to applied auxin. Perhaps these changes are also important in normal gravitropism.     

The binding of molybdate to uteroferrin. Hyperfine interactions of the binuclear center with 95Mo, 1H, and 2H

Uteroferrin, an acid phosphatase with a spin-coupled and redox-active binuclear iron center, is paramagnetic in its pink, enzymatically active, mixed-valence (S = 1/2) state. Phosphate, a product and inhibitor of the enzymatic activity of uteroferrin, converts the pink, EPR-active form of the protein to a purple, EPR-silent species. In contrast, molybdate, a tetrahedral oxyanion analog of phosphate, transforms the EPR spectrum of uteroferrin from a rhombic to an axial form. With both electron spin echo envelope modulation (ESEEM) and electron nuclear double resonance (ENDOR) spectroscopies, we observe a hyperfine interaction of [95Mo]molybdate with the S = 1/2, Fe(II)-Fe(III) center of the protein. A pair of 95Mo resonances centered at the 95Mo Larmor frequency at the applied magnetic field and separated by a hyperfine coupling constant of 1.2 MHz is evident. Therefore, a single monomeric species of molybdate is close to, and likely a ligand of, the binuclear cluster. 1H ENDOR studies on uteroferrin reveal at least six sets of lines mirrored about the 1H Larmor frequency. Two pairs of these lines become reduced in intensity when the protein is exchanged against D2O. Moreover, ESEEM and 2H ENDOR spectra display resonances at the 2H Larmor frequency. Therefore, the metal-binding region of the protein is accessible to solvent. Additional deuterium lines observable by ESEEM spectroscopy provide evidence for a population of strongly coupled, readily exchangeable protons associated with the binuclear center. The measured hyperfine coupling constants for these deuterons are orientation-dependent with splittings of nearly 4 MHz at g3 = 1.59 and less than 1 MHz at g1 = 1.94. In the presence of molybdate, ESEEM spectra of D2O-exchanged samples reveal a resonance at the 2H Larmor frequency, with no evidence of spectral components due to strongly coupled deuterons. 1H ENDOR studies of the uteroferrin-molybdate complex show at least seven pairs of lines, mirrored about the 1H Larmor frequency, of which one pair becomes attenuated in amplitude upon deuteration. The active site thus remains accessible to solvent in the presence of molybdate.