Identification of Synapse Specific Components: Synaptic Glycoproteins, Proteins, and Transmitter Binding Sites

Synaptic junctions (SJ) were prepared from synaptic plasma membranes (SPM) by extraction with Triton X-100 and density gradient centrifugation. These SJs were enriched in certain Concanavalin A (Con A) binding glycoproteins, the 52,000 Mr postsynaptic density (PSD) protein, and receptor sites for L-glutamate, L-aspartate, kainic acid (KA) but not quinuclidinyl benzilate (QNB). Various other membrane fractions were extracted by means of the same procedure. Those fractions prepared from light SPMs and crude myelin contained identifiable synaptic junctions and were also highly enriched in the synaptic components. The SJ-like fraction from mitochondria did not contain any of the characteristic synaptic macromolecules. However, this fraction from microsomes contained levels of the 52,000 Mr PSD protein and binding sites for L-glutamate (L-Glu) and L-aspartate (L-Asp) similar to true synaptic junctions, although the Con A binding glycoproteins and KA binding sites were nearly absent. On the basis of electron microscopy, the SJ-like fraction from microsomes did not contain structures recognizable as SJs. Thus, the Con A binding glycoproteins and KA binding sites appear to be excellent markers for the SJ.     

Distribution of [3H]Dihydrotetrabenazine Binding in Bovine Striatal Subsynaptic Fractions: Enrichment of Higher Affinity Binding in a Synaptic Vesicle Fraction

[3H]Dihydrotetrabenazine bound to a single class of binding sites in bovine striatal synaptic vesicles with an apparent dissociation constant of 3-9 nM. This is comparable to the inhibitory potency of dihydrotetrabenazine in catecholamine transport assays. In contrast to these results, [3H]dihydrotetrabenazine bound to at least two classes of sites in all other subsynaptic fractions investigated. The higher affinity class of sites was comparable in affinity to that of synaptic vesicles, whereas the lower affinity sites exhibited an apparent dissociation constant of 95-400 nM. Higher affinity sites were most abundant in the synaptic vesicle fraction, and little higher affinity binding was observed in mitochondrial and myelin fractions, or in highly purified synaptic plasma membranes. Lower affinity binding was not enriched in any subsynaptic fraction and was the only class of binding sites detected in homogenates of liver and diaphragm. The distribution of the presynaptic vesicle marker synaptophysin corresponded with that of higher affinity but not lower affinity binding. These results are consistent with the expectation that the higher affinity sites are associated primarily with synaptic vesicles and other neuronal entities that are in communication with these organelles.     

Characterization of L-Glutamate Binding Sites in Rat Spinal Cord Synaptic Membranes: Evidence for Multiple Chloride Ion-Dependent Sites

The effects of various ions on L-glutamate (L-Glu) binding sites (Na+-dependent, Cl(-)-dependent, and Cl(-)-independent) in synaptic plasma membranes (SPM) isolated from rat spinal cord and forebrain were examined. Cl(-)-dependent binding sites were over twofold higher in spinal cord (Bmax = 152 +/- 34 pmol/mg protein) as compared to forebrain SPM (Bmax = 64 +/- 12 pmol/mg protein). Na+-dependent binding, on the other hand, was nearly sixfold less in spinal cord (Bmax = 74 +/- 10 pmol/mg protein) compared to forebrain SPM (408 +/- 26 pmol/mg protein). Uptake of L-Glu (Na+-dependent) was also eightfold less in the P2 fraction from spinal cord relative to forebrain (Vmax of 2.89 and 22.3 pmol/mg protein/min, respectively). The effects of Na+, K+, NH4+, and Ca2+ on L-Glu binding sites were similar in both regions of the CNS. In addition, in spinal cord membranes, Br-, I-, and NO3- were equivalent to Cl- in their capacity to stimulate L-Glu binding, whereas F- and CO3- were less effective. Cl(-)-dependent L-Glu binding in spinal cord membranes consisted of two distinct sites. The predominant site (74% of the total) had characteristics similar to the Cl(-)-dependent binding site in forebrain membranes [i.e., Ki values of 5.7 +/- 1.4 microM and 119 +/- 38 nM for 2-amino-4-phosphonobutyric acid (AP4) and quisqualic acid, (QUIS), respectively]. The other Cl(-)-dependent site was unaffected by AP4 but was blocked by QUIS (Ki = 14.2 +/- 4.8 microM).     

Two mechanistically distinct forms of endocytosis in adrenal chromaffin cells: Differential effects of SH3 domains and amphiphysin antagonism

We previously identified two forms of endocytosis using capacitance measurements in chromaffin cells: rapid endocytosis (RE), dynamin-1 dependent but clathrin-independent and slow endocytosis (SE), dynamin-2 and clathrin-dependent. Various recombinant SH3 domains that interact with the proline-rich domain of dynamin were introduced into single cells via the patch pipette. GST-SH3 domains of amphiphysin-1, intersectin-IC, and endophilin-I inhibited SE but had no effect on RE. Grb2-SH3 (N-terminal) or a mutant of amphiphysin-1-SH3 was inactive on either process. These data confirm that dynamin-1 dependent RE is independent of clathrin and show that amphiphysin is exclusively associated with clathrin and dynamin-2-dependent SE.     

Altered Dynamics of S. aureus Phosphofructokinase via Bond Restraints at Two Distinct Allosteric Binding Sites

The effect of perturbation at the allosteric site was investigated through several replicas of molecular dynamics (MD) simulations conducted on bacterial phosphofructokinase (SaPFK). In our previous work, an alternative binding site was estimated to be allosteric in addition to the experimentally reported one. To highlight the effect of both allosteric sites on receptor’s dynamics, MD runs were carried out on apo forms with and without perturbation. Perturbation was achieved via incorporating multiple bond restraints for residue pairs located at the allosteric site. Restraints applied to the predicted site caused one dimer to stiffen, whereas an increase in mobility was detected in the same dimer when the experimentally resolved site was restrained. Fluctuations in C_α-C_α distances which is used to disclose residues with high potential of communication indicated a marked increase in signal transmission within each dimer as the receptor switched to a restrained state. Cross-correlation of positional fluctuations indicated an overall decrease in the magnitude of both positive and negative correlations when restraints were employed on the predicted allosteric site whereas an exact opposite effect was observed for the reported site. Finally, mutual correspondence between positional fluctuations noticeably increased with restraints on predicted allosteric site, whereas an opposite effect was observed for restraints applied on experimentally reported one. In view of these findings, it is clear that the perturbation of either one of two allosteric sites effected the dynamics of the receptor with a distinct and contrasting character.     

Two Distinct Calmodulin Binding Sites in the Third Intracellular Loop and Carboxyl Tail of Angiotensin II (AT1A) Receptor

In this study, we present data that support the presence of two distinct calmodulin binding sites within the angiotensin II receptor (AT_1A), at juxtamembrane regions of the N-terminus of the third intracellular loop (i3, amino acids 214–231) and carboxyl tail of the receptor (ct, 302–317). We used bioluminescence resonance energy transfer assays to document interactions of calmodulin with the AT_1A holo-receptor and GST-fusion protein pull-downs to demonstrate that i3 and ct interact with calmodulin in a Ca²⁺-dependent fashion. The former is a 1–12 motif and the latter belongs to 1-5-10 calmodulin binding motif. The apparent Kd of calmodulin for i3 is 177.0±9.1 nM, and for ct is 79.4±7.9 nM as assessed by dansyl-calmodulin fluorescence. Replacement of the tryptophan (W219) for alanine in i3, and phenylalanine (F309 or F313) for alanine in ct reduced their binding affinities for calmodulin, as predicted by computer docking simulations. Exogenously applied calmodulin attenuated interactions between G protein βγ subunits and i3 and ct, somewhat more so for ct than i3. Mutations W219A, F309A, and F313A did not alter Gβγ binding, but reduced the ability of calmodulin to compete with Gβγ, suggesting that calmodulin and Gβγ have overlapping, but not identical, binding requirements for i3 and ct. Calmodulin interference with the Gβγ binding to i3 and ct regions of the AT_1A receptor strongly suggests that calmodulin plays critical roles in regulating Gβγ-dependent signaling of the receptor.     

Identification of Two APOBEC3F Splice Variants Displaying HIV-1 Antiviral Activity and Contrasting Sensitivity to Vif

Approximately half of all human genes undergo alternative mRNA splicing. This process often yields homologous gene products exhibiting diverse functions. Alternative splicing of APOBEC3G (A3G) and APOBEC3F (A3F), the major host resistance factors targeted by the HIV-1 protein Vif, has not been explored. We investigated the effects of alternative splicing on A3G/A3F gene expression and antiviral activity. Three alternatively spliced A3G mRNAs and two alternatively spliced A3F mRNAs were detected in peripheral blood mononuclear cells in each of 10 uninfected, healthy donors. Expression of these splice variants was altered in different cell subsets and in response to cellular stimulation. Alternatively spliced A3G variants were insensitive to degradation by Vif but displayed no antiviral activity against HIV-1. Conversely, alternative splicing of A3F produced a 37-kDa variant lacking exon 2 (A3FΔ2) that was prominently expressed in macrophages and monocytes and was resistant to Vif-mediated degradation. Alternative splicing also produced a 24-kDa variant of A3F lacking exons 2–4 (A3FΔ2–4) that was highly sensitive to Vif. Both A3FΔ2 and A3FΔ2–4 displayed reduced cytidine deaminase activity and moderate antiviral activity. These alternatively spliced A3F gene products, particularly A3FΔ2, were incorporated into HIV virions, albeit at levels less than wild-type A3F. Thus, alternative splicing of A3F mRNA generates truncated antiviral proteins that differ sharply in their sensitivity to Vif.     

Binding and incorporation of lecithin-cholesterol vesicles to lymphocytes: A spin-label study

Summary When lecithin-cholesterol vesicles, containing the membrane-bound spin probe 3-doxyl-cholestane, were set in contact with mouse lymphocytes, the vesicles adsorbed to the cell and vesicle-membrane components were transferred to it. The spin probe was enzymatically reduced at the inside of the cell membrane. The spin-label method provided a means to determine quantitatively the extent of vesicles adsorption and vesicle-cell fusion by measuring the transfer of vesicles membrane material to the cell. This method, together with the reduction of spin label by the cell, allowed also a quantitative estimate of the extent of endocytosis during cell-liposome interaction.     

Identification of Novel Crk-associated Substrate (p130Cas) Variants with Functionally Distinct Focal Adhesion Kinase Binding Activities

Elevated levels of p130^Cas (Crk-associated substrate)/BCAR1 (breast cancer antiestrogen resistance 1 gene) are associated with aggressiveness of breast tumors. Following phosphorylation of its substrate domain, p130^Cas promotes the integration of protein complexes involved in multiple signaling pathways and mediates cell proliferation, adhesion, and migration. In addition to the known BCAR1-1A (wild-type) and 1C variants, we identified four novel BCAR1 mRNA variants, generated by alternative first exon usage (1B, 1B1, 1D, and 1E). Exons 1A and 1C encode for four amino acids (aa), whereas 1D and 1E encode for 22 aa and 1B1 encodes for 50 aa. Exon 1B is non-coding, resulting in a truncated p130^Cas protein (Cas1B). BCAR1-1A, 1B1, and variant 1C mRNAs were ubiquitously expressed in cell lines and a survey of human tissues, whereas 1B, 1D, and 1E expression was more restricted. Reconstitution of all isoforms except for 1B in p130^Cas-deficient murine fibroblasts induced lamellipodia formation and membrane ruffling, which was unrelated to the substrate domain phosphorylation status. The longer isoforms exhibited increased binding to focal adhesion kinase (FAK), a molecule important for migration and adhesion. The shorter 1B isoform exhibited diminished FAK binding activity and significantly reduced migration and invasion. In contrast, the longest variant 1B1 established the most efficient FAK binding and greatly enhanced migration. Our results indicate that the p130^Cas exon 1 variants display altered functional properties. The truncated variant 1B and the longer isoform 1B1 may contribute to the diverse effects of p130^Cas on cell biology and therefore will be the target of future studies.     

Glycine Binding to Rat Cortex and Spinal Cord: Binding Characteristics and Pharmacology Reveal Distinct Populations of Sites

Glycine is the principal inhibitory neurotransmitter in posterior regions of the brain. In addition, glycine serves as an allosteric regulator of excitatory neurotransmission mediated by the N-methyl-D-aspartate (NMDA) acidic amino acid receptor subtype. The studies presented here characterize [3H]glycine binding to washed membranes prepared from rat spinal cord and cortex, areas enriched in glycine inhibitory and NMDA receptors, respectively, in an attempt to define the glycine recognition sites on the two classes of receptors. Specific binding for [3H]glycine was seen in both cortex and spinal cord. Saturation analyses in cortex were best fitted by a two-site model with respective equilibrium dissociation constants (KD values) of 0.24 and 5.6 microM and respective maximal binding constants (Bmax values) of 3.4 and 26.7 pmol/mg of protein. Similar analyses in spinal cord were best fitted by a one-site model with a KD of 5.8 microM and Bmax of 20.2 pmol/mg of protein. Na+ had no effect on [3H]glycine binding to cortical membranes but increased the binding to spinal cord membranes by greater than 15-fold. This Na+-dependent binding may reflect glycine binding to the recognition site of the high-affinity, Na+-dependent glycine uptake system. Several short-chain, neutral amino acids displaced [3H]glycine binding from both cortical and spinal cord membranes. The most potent displacers of [3H]glycine binding to cortical membranes were D-serine and D-alanine, followed by the L-isomers of serine and alanine and beta-alanine. In contrast, D-serine and D-alanine were similar in potency to L-serine in spinal cord membranes. Compounds active at receptors for the acidic amino acids had disparate effects on the binding of [3H]glycine. At 10 microM, NMDA resulted in a 25% increase, whereas D- and L-2-amino-5-phosphonovaleric acid at 100 microM resulted in a 30% decrease, in [3H]glycine binding to cortical membranes. Kynurenic acid was the most potent of the acidic amino acid-related compounds at displacing [3H]glycine binding. In cortical membranes, kynurenic acid displacement was resolved into a high- and a low-affinity component; the high-affinity component displaced the high-affinity component of [3H]glycine binding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stress Upregulates TPH1 but not TPH2 mRNA in the Rat Dorsal Raphe Nucleus: Identification of Two TPH2 mRNA Splice Variants

Serotonin is implicated in stress-related psychopathologies. Two isoforms of the rate-limiting enzyme of serotonin biosynthesis, tryptophan hydroxylase, TPH1 and TPH2, are known. We show here that in the rat dorsal raphe nucleus (DRN), the nucleus that contains the highest number of 5-HT neurons in the brain, TPH1 mRNA reveals a low level of expression but is detectable both by quantitative real-time PCR and in situ hybridization whereas in the pineal gland (PiG), TPH1 mRNA is strongly expressed. To examine effects of stress on TPH expression we exposed male Wistar rats to daily restraint stress for 1 week. As shown by quantitative real-time PCR, TPH1 mRNA is 2.5-fold upregulated by the stress in DRN but not in PiG. Using 3′-RACE, we identified two TPH2 mRNA splice variants in the rat DRN which differ in the length of their 3′-untranslated regions (UTRs). TPH2b (with a short 3′-UTR) is the predominant variant in the DRN, whereas TPH2a (with a longer 3′-UTR) shows a low abundance in this nucleus. In the PiG, only TPH2b is detectable revealing a low level of expression. Expression of both TPH2 splice variants is not affected by stress, neither in DRN nor in the PiG. These data indicate that TPH1 in the serotonergic neurons of the DRN might be relevant for stress-induced psychopathologies.     

Developmental Changes in the Differential Expression of Two Serotonin 5-HT3 Receptor Splice Variants in the Rat

Abstract: PCR was used to isolate identical partial cDNA clones encoding a serotonin 5-HT₃ receptor subunit from rat nodose and superior cervical ganglia. The amino acid sequence predicted from these clones, extending from the putative transmembrane domain I to the stop codon, demonstrated a 93% homology with the 5-HT₃ receptor A (R-A) subunit cloned from NCB 20 hybridoma mouse neuroblastoma/Chinese hamster embryonic brain cells. Comparison of the sequences of the rat gene and cDNA encoding this subunit revealed a five amino acid deletion, GSLLP, located within the putative second intracellular loop of the receptor subunit. This deletion was shown to occur at an intron/exon junction. Therefore, alternative splicing was probably responsible for the presence of short (5-HT₃ R-A_S) and long (5-HT₃ R-A_L) forms of 5-HT₃ R-A mRNA in these ganglia. PCR experiments, with specific primers located upstream and downstream of the GSLLP deletion, were used to detect reverse transcribed 5-HT₃ R-A mRNAs. A short fragment (92 bp), corresponding to the deleted form, and a long fragment (107 bp), corresponding to the nondeleted form, were amplified from various regions of the CNS and peripheral ganglia of the rat, as well as from NG108-15 hybridoma cells. In the adult rat, the ratio of the two forms varied very little from one tissue to another, the long form corresponding to only ～10% of the total 5-HT₃ R-A mRNA. Study of their respective distributions during ontogeny demonstrated a differential expression of the short and long forms in some tissues during late embryonic development, at embryonic day 17 (E17) or E20. In particular, the long form amounted to about one-third of the total 5-HT₃ R-A mRNA in the cerebral cortex and hippocampus at E17, and this proportion reached 50 and 75% in the superior cervical ganglion and nodose ganglion, respectively, at E20. These data indicate that alternative splicing of the 5-HT₃ R-A mRNA is regulated in the CNS and PNS during development in the rat.     

Identification of ω-Conotoxin Binding Sites on Adrenal Medullary Membranes: Possibility of Multiple Calcium Channels in Chromaffin Cells

Binding of 125I-omega-conotoxin GVIA and [3H]nitrendipine to membranes from bovine adrenal medulla was investigated to test for the presence of N- and L-type Ca2+ channels in adrenal chromaffin cells. Saturable, high-affinity binding sites for 125I-omega-conotoxin and [3H]nitrendipine were detected in a membrane fraction from adrenal medulla. [3H]Nitrendipine binding sites were found to have a KD of 500 +/- 170 pM and a Bmax of 26 +/- 11 pmol/g of protein. 125I-omega-Conotoxin binding sites had a KD of 215 +/- 56 pM and a Bmax of 105 +/- 18 pmol/g of protein, about four times the number of sites found for [3H]nitrendipine. 125I-omega-Conotoxin binding was potently inhibited by unlabeled toxin and Ca2+ but was unaffected by dihydropyridines, verapamil, and diltiazem. [3H]Nitrendipine binding was not affected by omega-conotoxin, whereas it was inhibited by other dihydropyridines. Bay K 8644 potentiated K+-evoked cytosolic Ca2+ transients measured by fura-2 fluorescence, and this potentiation was completely blocked by nifedipine. In contrast, omega-conotoxin had no effect on Bay K 8644-evoked Ca2+ transients. Thus, the binding sites for omega-conotoxin and for nitrendipine appear to be different. The results confirm the presence of L-type Ca2+ channels and open the possibility of N-type Ca2+ channels as the omega-conotoxin binding sites in chromaffin cell membranes.     

RGSZ1 and Ret RGS: Two of Several Splice Variants from the Gene RGS20

RGSZ1 and Ret RGS, members of the regulator of G-protein signaling (RGS) family, are GTPase-activating proteins (GAPs) with high selectivity for G alpha(z). We show here that RGSZ1 and Ret RGSZ1 are products of two of several splice variants of one gene, RGS20. RGS20 spans approximately 107 kb and contains at least seven exons. Five exons account for RGSZ1, including a single exon distinct to RGSZ1 that encodes a newly identified amino-terminal region. The previously described open reading frame (ORF) and 3' untranslated region are encoded by four downstream exons that also encode about half of Ret RGS. The 5' end of the RGSZ1 ORF contains several in-frame ATG codons (3-5 depending on the species), and multiple translational start sites may help explain the molecular weight heterogeneity of purified bovine brain RGSZ. Ret RGS replaces the 24 N-terminal amino acid residues of RGSZ1 with a large, N-terminal region that initially distinguished the bovine Ret RGS from human and mouse RGSZ1. This N-terminal domain is encoded by two distinct 5' exons that are variably combined with the four downstream exons shared with RGSZ1 to produce at least six mRNAs. They encode proteins with N termini that vary in size, hydrophobicity, and the presence of a cysteine string. At least two mRNAs that include the exon that encodes the N-terminal region unique to RGSZ1 were found in brain and a few other tissues, but not retina. RGS20 thus can account for multiple G(z)-selective GAPs in different tissues.     

Tubulin Stability and Decay: Mediation by Two Distinct Classes of IKP104-Binding Sites

IKP104, a novel antimitotic drug, has two classes of binding sites on bovine brain tubulin with different affinities. IKP104, by itself, enhances the decay of tubulin, but in the presence of colchicine or podophyllotoxin, it stabilizes tubulin instead of opening up the hydrophobic areas [Luduena et al. (1995), Biochemistry 34, 15751–15759], Here, we have dissected these two apparently contradictory effects of IKP104 by cleaving the C-terminal ends of both and subunits of tubulin with subtilisin. We have found that the selective removal of the C-terminal ends from both the and subunits of tubulin lowers the sulfhydryl titer by approximately 1.5 mol/mol of dimer. Interestingly, IKP104 does not increase either the sulfhydryl liter or the exposure of hydrophobic areas of this subtilisin-treated tubulin (_ss). Moreover, IKP104 lowers the sulfhydryl titer of _ss tubulin approximately by 1 mol/mol and appears to inhibit completely the time-dependent decay of _ss tubulin. The cleavage at the C-terminal ends of both and modulates the effect of IKP104 on the subunit, but not on the subunit. Fluorometric binding data analysis suggests that IKP104 binds to the _ss tubulin only at the high-affinity site; the low-affinity site(s) disappear almost completely. The sulfhydryl titer data for and and the fluoromelric data therefore suggest that the interaction of IKP104 at the high-affinity site on tubulin is not regulated by the C-terminal domains of and and the effect of the high-affinity site is restricted largely to the subunit, while the low-affinity-site binding is modulated by the C-terminal domain of . It also appears that the stabilization and the acceleration of the decay of tubulin are mediated by distinct interactions of IKP104 with its high- and low-affinity sites on tubulin, respectively.     

Interaction of Opiates with Opioid Binding Sites in the Bovine Adrenal Medulla: II. Interaction with K Sites

Abstract: In this study we examined the interaction of opiates with K binding sites in the bovine adrenal medulla. [³H]Ethylketocyclazocine (EKC), [³H]etorphine, and [³H]bremazocine stereoselective bindings were used to assay these interactions. The K sites were found to be heterogeneous: [³H]bremazocine identified with high affinity all subtypes of these sites. [³H]EKC, in the presence of saturating concentrations of [D-Ala², D-Leut]-enkephalin (DADLE) (5μM), was used to identify K¹ sites, on which dynorphin A (1–13) bound with high affinity. Either [³H]EKC or [³H]etorphine in the presence of 5μM DADLE identified the K² subtype. This subtype was found to interact with β-endorphin and especially with the octapeptide Met⁵-enkephalyl-Arg⁶-Gly⁷-Leu⁸. Furthermore, [³H]etorphine identified in the bovine adrenal medulla a third high-affinity component, in the presence of 5 μM DADLE. This residual interaction was found to be equally stereoselective and presenting K selectivity. Met⁵-enkephalyl-Arg⁶-Phe⁷ interacted preferentially with this site. The three K subtypes interacted differentially with monovalent (Na⁺, K⁺, and Li⁺) and divalent (Ca²⁺, Mg²⁺, and Mn²⁺) ions by modification of the apparent concentration of the accessible sites and/or by changes of the apparent K_D for radioligands. Modifying agents (proteolytic enzymes, thiol-modifying reagents, and A₂-phospholipase) produced different effects on each subtype of the K site, suggesting a different protein (or protein-lipid?) composition.     

Identification of Multiple Binding Sites for [3H]5-Hydroxytryptamine in the Rat CNS

Recent studies indicate that there may be multiple subtypes of [3H]5-hydroxytryptamine ([3H]5-HT) binding sites. Mianserin and spiperone inhibited the specific binding of [3H]5-HT (2-3 nM) to rat brain cortical membranes with shallow displacement curves. The displacement data for spiperone were best described by the presence of three independent binding sites, for which spiperone had high, medium, and low affinities. The displacement data for mianserin were best fitted by two independent, high- and low-affinity sites. The inclusion of mianserin (250 nM) to inhibit [3H]5-HT binding to the mianserin-sensitive site selectively blocked one of the sites discriminated by spiperone. These results suggest the presence of three binding sites for [3H]5-HT, one blocked by low concentrations of spiperone (5-HT1A), one blocked by low concentrations of mianserin (5-HT1C), and one blocked only by high concentrations of both mianserin and spiperone (5-HT1B). Regional differences in the relative densities of the three sites were observed. The hippocampus was rich in 5-HT1A sites, whereas the striatum contained mainly 5-HT1B and 5-HT1C sites. Selective degeneration of 5-HT-containing nerve terminals induced by the neurotoxin 5,7-dihydroxytryptamine increased binding to all three sites in the cerebral cortex. Binding of [3H]5-HT to the three sites was differentially modulated by CaCl2 and guanylimidodiphosphate. The present data suggest the presence of three independent 5-HT1 binding sites having different affinities for mianserin and spiperone and having different regional distributions.     

Two Distinct Populations of Primary Cytotoxic Cells Infiltrating into Allografted Tumor Rejection Sites: Infiltration of Macrophages Cytotoxic against Allografted Tumor Precedes That of Multiple Sets of Cytotoxic T Lymphocytes with Distinct Specificity to Alloantigens

It has been reported that the rejection of tumor allografts is mainly mediated by cytotoxic T lymphocytes (CTLs). Here, we characterized the cytotoxic effector cells of C57BL/6 (B6; H-2^b) mice infiltrating into the rejection site of the i.p. allografted Meth A fibrosarcoma (or P815 mastocytoma) cells of H-2^d origin. Two types of cytotoxic cells (i.e., CD8⁺ CTLs and macrophages (Mφs)) were identified by flow cytometric fractionation of the infiltrates or by specific in vitro elimination of cells either with antibody (Ab)-coated beads or with an Ab-plus complement. Of particular interest, these effector cells showed distinct and unique target specificities. First, the CTLs were inactive against transplanted tumor (e.g., Meth A) cells, whereas they were cytotoxic against donor-related concanavalin A (Con A) blasts as well as CTLL-2 (H-2^b) cells transfected with a class I gene of H-2^d origin. A cold target competition assay suggested that the CTLs were composed of multiple sets of T cells, each of which specifically recognized different allo-antigens. Second, the Mφs lysed the allografted tumor cells but were inert toward the Con A blasts and the CTLL-2 transfectants. Unexpectedly, the infiltration of Mφs preceded the infiltration of CTLs by several days during the course of rejection. These results indicate that two distinct populations of unique cytotoxic cells (i.e., CTLs and Mφs) are induced in the allografted tumor rejection site, and that the infiltration of cytotoxic Mφs responsible for rejection precedes that of the CTLs cytotoxic against cells expressing donor-related allo-antigens.     

Identification of the Binding Site of Methylglyoxal on Glutathione Peroxidase: Methylglyoxal Inhibits Glutathione Peroxidase Activity via Binding to Glutathione Binding Sites Arg 184 and 185

Methylglyoxal (MG), a physiological &#102 -dicarbonyl compound is derived from glycolytic intermediates and produced during the Maillard reaction. The Maillard reaction, a non-enzymatic reaction of ketones and aldehydes with amino group of proteins, contributes to the aging of proteins and to complications associated with diabetes. In our previous studies (Che, et al. (1997) "Selective induction of heparin-binding epidermal growth factor-like growth factor by MG and 3-deoxyglucosone in rat aortic smooth muscle cells. The involvement of reactive oxygen species formation and a possible implication for atherogenesis in diabetes". J. Biol. Chem., 272 , 18453-18459), we reported that MG elevates intracellular peroxide levels, but the mechanisms for this remain unclear. Here, we report that MG inactivates bovine glutathione peroxidase (GPx), a major antioxidant enzyme, in a dose- and time-dependent manner. The use of BIAM labeling, it was showed that the selenocysteine residue in the active site was intact when GPx was incubated with MG. MALDI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) and protein sequencing examined the possibility that MG modifies arginine residues in GPx. The results show that Arg 184 and Arg 185, located in the glutathione binding site of GPx was irreversively modified by treatment with MG. Reactive dicarbonyl compounds such as 3-deoxyglucosone, glyoxal and phenylglyoxal also inactivated GPx, although the rates for this inactivation varied widely. These data suggest that dicarbonyl compounds are able to directly inactivate GPx, resulting in an increase in intracellular peroxides which are responsible for oxidative cellular damage.