A functional study of proximal goat ß-casein promoter and intron 1 in immortalized goat mammary epithelial cells.

Goat ß-casein (CSN2) promoter has been extensively used to derive expression of recombinant therapeutic protein in transgenic goats; however, little direct evidence exists for signaling molecules and the cis-elements of goat CSN2 promoter in response to lactogenic hormone stimulation in goat mammary epithelial cells. Here, we use an immortalized caprine mammary epithelial cell line (CMC) to search for evidence of the above. Serial 5'-flanking regions deleted of promoter and intron 1 in goat CSN2 (-4,047 to +2,054) driven by firefly luciferase reporter gene were constructed and applied to measure promoter activity in CMC. The intron 1 region (+393 to +501) significantly decreased basal activity of the promoter. This finding contradicts other studies of the role of intron 1. The signal transducer and activator of transcription (STAT)5a played a significant role in activating promoter activity by prolactin stimulation. Hydrocortisone enhanced and prolonged the activity of STAT5a and promoter in CMC, but was independent of the glucocorticoid receptor response element. The minimum length of the CSN2 promoter segment in response to lactogenic stimulation was confirmed by 5' serial deletions. A cis-element located from -300 to -90 in proximal goat CSN2 promoter that is absent in bovine and human CSN2 promoter was newly identified. We demonstrated the presence of a STAT5a binding site (-102 to -82) and preservation of the guanosine nucleotide at position -90 based on responses to the presence of lactogenic hormone using internal deletions and point mutations of the predicted STAT5a binding site, and chromatin immunoprecipitation assay. Together, these findings demonstrate that the proximal -300 bp of goat CSN2 promoter containing the STAT5a binding site (-102 to -82) is the response element for lactogenic hormone stimulation. Additionally, intron 1 may be required for tissue or developmental stage-specific expression in mammary gland. The role of the far-distal regions of goat CSN2 promoter in high-level lactogenic hormone induction and specific expression require further examination.

Whole exome sequencing identifies a novel mutation in the transglutaminase 6 gene for spinocerebellar ataxia in a Chinese family.

Autosomal dominant spinocerebellar ataxias (SCA) constitute a heterogeneous group of inherited disorders. The transglutaminase 6 (TGM6) gene was recently suggested as a SCA causative gene in Chinese SCA families. In this study, two affected members of a three-generation Chinese family with SCA characterized by progressive cerebellar ataxia and lower limb pyramidal signs were subjected to whole exome sequencing. Through bioinformatics analysis of the sequence variants in these two individuals, we identified a novel mutation in the TGM6 gene (c.1528G>C) which showed perfect co-segregation with disease phenotype in all nine members of this family. This finding confirms that mutations in TGM6 gene represent an important cause of SCA in Chinese. This study also shows that whole exome sequencing of a small number of affected individuals, leveraged on bioinformatics analysis, can be an efficient strategy for identifying causative mutations in rare Mendelian disorders.

Neuromyelitis optica-IgG in idiopathic inflammatory demyelinating disorders amongst Hong Kong Chinese.

BACKGROUND: Idiopathic inflammatory demyelinating disorders (IIDD) affect the central nervous system. In classical multiple sclerosis (CMS), brain, optic nerves [optic neuritis (ON)] and spinal cord [acute transverse myelitis (ATM)] are affected. In neuromyelitis optica (NMO), optic nerves and spinal cord are predominantly affected. NMO-IgG, an autoantibody targeting aquaporin-4, is a marker for NMO. We studied the frequency and clinical relevance of NMO-IgG seropositivity in IIDD patients. METHODS: Neuromyelitis optica-IgG was detected by indirect immunofluorescence using primate cerebellum. RESULTS: Neuromyelitis optica-IgG was detected in six of 10 NMO patients (60%), six of 10 idiopathic relapsing transverse myelitis (IRTM) patients (60%), two of nine idiopathic relapsing ON patients (22%), one of 11 patients (9%) having single ON attack, one of 30 CMS patients (3%), and none of patients having single ATM attack or controls. Comparing NMO-IgG seropositive (n = 12) with NMO-IgG seronegative (n = 8) patients having NMO or IRTM, NMO-IgG seropositivity was associated with a higher relapse rate in first 2 years, 1.5 and 0.6 attacks/year for seropositive and seronegative groups respectively (P = 0.006), and non-significant trend towards more severe ON and myelitis with poorer clinical outcome. CONCLUSION: Neuromyelitis optica -IgG facilitates diagnosis of NMO spectrum disorders. NMO-IgG seropositivity is associated with higher relapse rate in first 2 years.

Effects of plasticisers and related compounds on the expression of the soluble form of catechol-O-methyltransferase in MCF-7 cells.

Previously we have shown that E2 down regulates S-COMT expression. Here the effects of four phthalate esters and 4-(tert-octyl)phenol on the intra-cellular levels of S-COMT and COMT activity were studied in MCF-7 cells as a measure of estrogenic activity of these compounds. The four phthalate esters caused significant reductions in both S-COMT protein and COMT activity levels. These effects were inhibited by the ERalpha receptor antagonist ICI182780. 4-(tert-octyl)phenol also caused reductions in these parameters, but the effects were not abolished by ICI182780. Assay of S-COMT protein levels represents a simple and convenient method of assessing the estrogenic potential of a compound.

Estrogenic phenol and catechol metabolites of PCBs modulate catechol-O-methyltransferase expression via the estrogen receptor: potential contribution to cancer risk.

Commercial PCB mixtures have been shown to induce liver tumors in female rats and this effect has been attributed to the effects of PCBs on estrogen metabolism. Catechol metabolites of PCBs are potent inhibitors of COMT activity and are likely to contribute significantly to reduced clearance of genotoxic catechol metabolites of estrogen. The effect of PCB metabolites on COMT expression in cultured cells was investigated to explore potential mechanisms by which PCB exposure alters catechol estrogen clearance. We hypothesize that estrogenic PCB metabolites may contribute to reduction of COMT expression via interaction with the estrogen receptor. To test this hypothesis, human MCF-7 cells were exposed to PCB analogues and the expression of COMT determined. Western blot analysis demonstrated that COMT protein levels were statistically significantly reduced by both the phenolic and the catechol compounds, an effect which was abolished by the anti-estrogen, ICI182780. The above suggests that COMT levels may be reduced by estrogenic PCB metabolites, via interactions between PCB metabolites and the ER. It supports the hypothesis that both phenolic and catechol metabolites of PCBs may contribute to PCB-mediated carcinogenesis through reduction of COMT levels and activities and subsequent reduction in clearance of endogenous and xenobiotic catechols.

Action of partially thiolated polynucleotides on the DNA polymerase alpha from regenerating rat liver.

The effects of partially thiolated polynucleotides on the DNA polymerase alpha from regenerating rat liver were investigated. The enzyme was isolated from the nuclear fraction essentially according to the method of Baril et al.; it was characterized as the alpha polymerase on the basis of its response to synthetic templates and its inhibition with N-ethylmaleimide. Although polycytidylic acid had no effect on the DNA polymerase alpha either as a template or as an inhibitor, partially thiolated polycytidylic acid (MPC) was found to be a potent inhibitor, its activity being directly related to its extent of thiolation (percentage of 5-mercaptocytidylate units in the polymer). In comparison, the DNA polymerase beta which was purified from normal rat liver nuclear fraction, was much less sensitive to inhibition by MPC. Analysis of the inhibition of the alpha polymerase by the method of Lineweaver and Burk showed that the inhibitory action of MPC was competitively reversible with the DNA template, but the binding of the 7.2%-thiolated MPC to the enzyme was much stronger than that of the template (Ki/Km less than 0.03). Polyuridylic acid as such showed some inhibitory activity which increased on partial thiolation, but the 8.4%-thiolated polyuridylic acid was less active than the 7.2% MPC. When MPC was annealed with polyinosinic acid, it lost 80% of its inhibitory activity in the double-stranded configuration. However, 1 to 2%-thiolated DNA isolates were significantly more potent inhibitors than were comparable (1.2%-thiolated) MPC and showed competitive reversibility with the unmodified (but "activated") DNA template. These results indicate that the inhibitory activities of partially thiolated polynucleotides depend not only on the percentage of 5-mercapto groups but also on the configuration, base composition, and other specific structural properties.

Plasma clearance and tissue distribution of partially thiolated polycytidylic acid and its degradation products in rodents.

Radioactive (35S-labeled) partially thiolated polycytidylic acid (MPC) was administered i.v. to male Sprague-Dawley rats. Blood samples were taken at various intervals, and the radioactivity in plasma was determined. The concentration of total radioactivity in plasma decreased rapidly postinjection, independently of the dose, and could not be readily resolved into a series of exponential terms with a high degree of confidence. Coadministration with polyinosinic acid in a 1:1 ratio significantly decreased the clearance of radioactive compounds from the plasma; moreover, the clearance of radioactivity decreased with increasing dose. Complexing with polyinosinic acid also decreased the rate of degradation of [35S]MPC as evidenced by an increase of the trichloroacetic acid-precipitable fraction (i.e., oligonucleotides larger than five to ten nucleotide units), from 0.45 to 0.92 of the total radioactivity in plasma 60 min postinjection. The plasma clearance and organ distribution of radioactivity following injection of [35S]MPC were determined in normal and leukemic RFM/Un mice. About 90% of the 35S radioactivity was removed from the plasma in 5 and 10 min, respectively, in these two groups of mice, and the residual plasma levels of radioactivity at any given time were twice as high in the leukemic group throughout an observation period of 1 hr. Organ distribution studies demonstrated significantly greater (per mg tissue) accumulation of radioactivity in the livers and spleens of the leukemic versus normal mice at all time points, while the corresponding data for the kidneys were similar for the two groups. Another study, comparing the radioactivity in suspended and washed spleen cells harvested 60 min postinjection, indicated that 4 to 10 times more MPC and/or 35S-labeled oligonucleotides were localized and bound intracellularly in the spleens of the leukemic mice. These studies of the pharmacokinetic properties and metabolic degradation of [35S]MPC suggest that this polynucleotide may be protected from degradation by complexing with polyinosinic acid and that preferential accumulation of [35S]MPC occurs in organs infiltrated by leukemic cells.

Hypofractionated radiation therapy for the treatment of microscopic canine soft tissue sarcoma.

Soft tissue sarcomas (STSs) are locally invasive and surgery with or without radiation therapy is the current standard of care in dogs. Typical protocols for treating incompletely excised STSs involve curative intent radiation with total dose in excess of 50 Gy. Forty-eight dogs with histologically confirmed incomplete or closely excised STSs were treated with a hypofractionated protocol that is typically reserved for palliative radiation therapy (RT) (6-8 Gy/weekly fractions to a total dose of 24-32 Gy). Ten dogs (21%) developed local recurrence, 11 dogs (23%) developed metastasis, and 3 dogs developed both (included in each group). The median progression free survival was 698 days. The local failure-free probability at 1 and 3 years was 81 and 73%. The 1 and 3 years tumour-specific overall survival was 81 and 61%. Long-term local tumour control was achieved in the majority of dogs. This protocol is reasonable to prescribe in older patients or when financial limitations exist.

Dopamine D2 long receptor-deficient mice display alterations in striatum-dependent functions.

The dopamine D2 receptor (D2) system has been implicated in several neurological and psychiatric disorders, such as schizophrenia and Parkinson's disease. There are two isoforms of the D2 receptor: the long form (D2L) and the short form (D2S). The two isoforms are generated by alternative splicing of the same gene and differ only by 29 amino acids in their protein structures. Little is known about the distinct functions of either D2 isoform, primarily because selective pharmacological agents are not available. We generated D2L receptor-deficient (D2L-/-) mice by making a subtle mutation in the D2 gene. D2L-/- mice (which still express functional D2S) displayed reduced levels of locomotion and rearing behavior. Interestingly, haloperidol produced significantly less catalepsy and inhibition of locomotor activity in D2L-/- mice. These findings suggest that D2L and D2S may contribute differentially to the regulation of certain motor functions and to the induction of the extrapyramidal side effects associated with the use of typical antipsychotic drugs (e.g., haloperidol). Quinpirole induced a similar initial suppression of locomotor activity in both D2L-/- and wild-type mice. In addition, the D2S receptor in the mutant mice functioned approximately equally well as did D2L as an impulse-modulating autoreceptor. This suggests that the functions of these two isoforms are not dependent on the formation of receptor heterodimers. Our findings may provide novel information for potentially developing improved antipsychotic drugs.

High-order fluorescence and exciton interaction in photosynthetic bacteria.

We have observed fluorescence at visible wavelengths from chromatophores of photosynthetic bacteria excited with infrared radiation which we attribute to bacteriochlorophyll of the antenna system. The fluorescence is prompt (no delay greater than 5 ns). Its spectrum shows peaks at 445, 530 (broad) and 600 nm when excited with either 694 or 868 nm. Quantum yield is of the order of 10(-9). The dependence on intensity indicates generation by mainly third-order processes which could involve triplet state in combination with excited singlets. Second-order single-singlet fusion could also contribute. The high-order fluorescence can also be explained as arising from absorption of a second photon by singlet excited states.

Expression of intracellular and GPI-anchored forms of GPI-specific phospholipase D in COS-1 cells.

Glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) is a secretory protein present in high amounts in mammalian body fluids. Its cDNA has been isolated and encodes a signal peptide of 23 amino acids and the mature protein of 816 amino acids. We generated cDNAs encoding a signal peptide-deficient and a GPI-anchored form of GPI-PLD and transiently transfected these constructs into COS-1 cells. The signal peptide-deficient form of GPI-PLD was expressed as a 90-kDa protein that was catalytically active and was localized intracellularly. Cells transfected with cDNA encoding the GPI-anchored form of GPI-PLD expressed a catalytically active enzyme of 100 kDa that could be labelled with [3H]ethanolamine demonstrating its modification by a GPI structure. Expression of the GPI-anchored form of GPI-PLD resulted in the release of endogenous GPI-anchored alkaline phosphatase from COS-1 cells, whereas expression of the intracellular form of GPI-PLD had no effect on membrane attachment of endogenous alkaline phosphatase. Similarly, in cells cotransfected with GPI-anchored placental alkaline phosphatase (PLAP) and the GPI-anchored form of GPI-PLD, PLAP was released into the cell culture supernatant while expression of the signal peptide-deficient form of GPI-PLD did not affect the amount of cell-associated PLAP.

Direct evidence of oxidized gold on supported gold catalysts.

Supported gold catalysts have drawn worldwide interest due to the novel properties and potential applications in industries. However, the origin of the catalytic activity in gold nanoparticles is still not well understood. In this study, time-of-flight secondary ion mass spectroscopy (TOF-SIMS) has been applied to investigate the nature of gold in Au (1.3 wt %)/gamma-Al2O3 and Au (2.8 wt %)/TiO2 catalysts prepared by the deposition-precipitation method. The SIMS spectrum of the supported gold catalysts presented AuO-, AuO2-, and AuOH- ion clusters. These measurements show direct evidence for oxidized gold on supported gold catalysts and may be helpful to gaining better understanding of the origin of the catalytic activity.

Desulfation of 3,5,3'-triiodothyronine sulfate by microsomes from human and rat tissues.

Subcellular preparations from rat liver, brain, and kidney and from human liver were tested for their ability to desulfate T3 sulfate (T3SO4). Activity was found associated with the microsomal fraction: rat liver was the most active, hydrolyzing 76 pmol/min.mg protein of T3SO4 while preparations from rat kidney and brain were about 1/5 and 1/20 as active. Microsomal preparations from human liver obtained at autopsy were as active as fresh rat preparations. Thyroxine sulfate was not an active substrate. Microsomes prepared with dithiothreitol and EDTA in order to detect deiodinating activity maintained T3SO4-desulfating activity. Cytosolic preparations containing arylsulfatase activities failed to desulfate T3SO4. Estrone sulfate, dehydroepiandrosterone sulfate, and nitrophenyl sulfate are known substrates for microsome-associated arylsulfatase activities, and these compounds were found to inhibit hydrolysis of T3SO4 to various extents. Of these competing sulfatase substrates, only dehydroepiandrosterone sulfate inhibits T3SO4 desulfation completely. In order to determine whether desulfation occurs in intact cells, isolated hepatocytes were incubated in the presence of 7 and 54 microM T3SO4. These cells were found to hydrolyze 1-1.5% of the sulfate ester/h for up to 3 h. The demonstration of this activity raises the possibility that these hepatic cells may be able to reactivate T3SO4, which has generally been regarded as an irreversibly inactivated metabolite.

Studies on the biological activity of triiodothyronine sulfate.

Hepatic microsomes and isolated hepatocytes in short term culture desulfate T3 sulfate (T3SO4). We, therefore, wished to determine whether T3SO4 could mimic the action of thyroid hormone in vitro. T3SO4 had no thyromimetic effect on the activity of Ca(2+)-ATPase in human erythrocyte membranes at doses up to 10,000 times the maximally effective dose of T3 (10(-10) mol/L). In GH4C1 pituitary cells, T3SO4 failed to displace [125I]T3 from nuclear receptors in intact cells or soluble preparations. Thus, T3SO4 was not directly thyromimetic in either an isolated human membrane system or a pituitary cell system in which nuclear receptor occupancy correlates with GH synthesis. Thyroid hormones inhibit [3H]glycosaminoglycan synthesis by cultured human dermal fibroblasts, and T3SO4 displayed about 0.5% the activity of T3 at 72 h. Human fibroblasts contained roughly the same level of microsomal p-nitrophenyl sulfatase activity as that previously observed in hepatic microsomes. Propylthiouracil (50 mumol/L) did not affect the action of T3SO4, suggesting that deiodination was not important for this activity of T3SO4. Thus, it appears T3SO4 has no intrinsic biological activity, but, under certain circumstances, may be reactivated by desulfation.

Addition of G418 and other aminoglycoside antibiotics to mammalian cells results in the release of GPI-anchored proteins.

Resistance to the neomycin analogue G418 forms the basis of a dominant marker selection system for mammalian cells transfected with the bacterial neomycin gene. We found that COS-1 cells stably transfected with the neomycin resistance gene had a greater than 50% reduction in cell-associated glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase (AP). A similarly reduced amount of AP was also observed in wild-type COS-1 cells incubated in the presence of G418 or other aminoglycoside antibiotics. The AP was released from cells into the culture supernatant in its GPI-anchored form. Our data suggest that the G418-induced reduction of AP involves a vesiculation process of COS-1 cells.

Lack of discrimination by agonists for D2 and D3 dopamine receptors.

The affinities of D3 dopamine receptors for antagonists are similar to those of D2 receptors. D3 receptors have been reported, however, to have affinities nearly 100-fold higher than those of D2 receptors for some agonists, including (+/-)-7-hydroxy-n,n-dipropyl-aminotetralin (7-OH-DPAT) and quinpirole. This has led to the use of these agonists to try to identify functional responses mediated by D3 receptors in vivo. However, D2 receptors exist in multiple states having high and low affinities for agonists. The G protein-coupled state of D2 receptors is believed to be the functional state of these receptors. When receptors were labeled with the D2 receptor antagonist [125I]-(S)-3-iodo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5,6- dimethoxysalicylamide ([125I]-NCQ-298) under conditions that promote uncoupling of receptors from G proteins, the affinities of D3 receptors were approximately 130-fold higher than those of D2 receptors for 7-OH-DPAT and quinpirole. When receptors were labeled with the D2 receptor agonist [125I]-(R)trans-7-hydroxy-2-[N-propyl-N-(3'-iodo-2'- propenyl)-amino]tetralin ([125I]-7-OH-PIPAT) under conditions that favor interactions of receptors with G proteins, the affinities of D3 receptors were less than sevenfold higher than the affinities of D2 receptors for the same drugs. Similarly, small differences in the affinities of D2 and D3 receptors for other agonists were seen when receptors were labeled with [125I]-7-OH-PIPAT. These data demonstrate that putative D3 receptor-selective agonists also interact with a high-affinity, G protein-coupled state of D2 receptors. The similarities in affinities of the agonist-preferring state of D2 and D3 receptors means that currently available agonists cannot be used to discriminate between behavioral effects mediated by D2 and D3 receptors.

"Experimentation" with chloroform.

A young patient presented with unconsciousness, cardiac arrhythmias, arterial hypotension, and mild intravascular hemolysis after intentional inhalation of chloroform. After the initial complications had resolved, nausea, loss of appetite, and mild transitory jaundice developed. Chloroform-associated hepatotoxicity was biochemically and histologically documented. Facilitating factors included long-term moderate alcohol consumption and an initial episode of arterial hypoxemia. Chloroform inhalation for recreational purposes is probably uncommon. Yet, because of its delayed onset, chloroform poisoning should be considered in acute liver disease without a clear antecedent cause.

Characterization of [125I]S(-)5-OH-PIPAT binding to dopamine D2-like receptors expressed in cell lines.

Recently, [125I]S(-)5-OH-PIPAT [5-hydroxy-2-(N-n-propyl-N-3'-iodo-2'-propenyl)aminotetralin], a derivative of S(-)5-OH-DPAT (5-hydroxy-N,N-di-n-propyl-aminotetralin), was reported to be a better radioiodinated dopamine D2-like receptor ligand than the previously reported iodinated ligand, [125I]R(+)7-OH-PIPAT. Therefore, in the present study, the binding profile of [125I]S(-)5-OH-PIPAT to D2-like receptors expressed in cell lines was established. High binding affinity (Kd = 0.3-0.4 nM) and NaCl sensitivity were displayed with this ligand in membranes of human embryonic kidney (HEK293) cells expressing either human D2 or rat D3 receptors and in Chinese hamster ovary (CHO) cells expressing human dopamine D4 receptors. Specific binding to D2 and D4 receptors was significantly increased in the presence of 2 mM MgCl2 and decreased in the presence of 100 microM 5'-guanylyl-imidodiphosphate (GMP-PNP). This finding is consistent with reports that 2-aminotetralin compounds display agonist properties. The specific binding to D3 receptors however, was not affected by either MgCl2 or GMP-PNP. This lack of GMP-PNP sensitivity for D3 receptors may result from inadequate G protein-receptor coupling in this cell line. The rank order of potency for inhibition of [125I]S(-)5-OH-PIPAT binding with various dopamine agents was consistent with reported values for D2, D3 and D4 receptors. In membranes prepared from Spodoptera frugiperda (Sf9) cells infected with baculovirus that contains DNA encoding D3 receptors, [125I]S(-)5-OH-PIPAT recognized only 70% of the receptor population labeled by [125I]NCQ298. This new ligand offers several unique advantages, including high specific activity, high binding affinity and selectivity for D2-like receptors, that make it an excellent probe for the investigation and the characterization of dopamine D2-like receptors.

Synthesis and optical resolution of (R)- and (S)-trans-7-Hydroxy-2-[N-propyl-N-(3'-iodo-2'-propenyl)amino]tetralin: a new D3 dopamine receptor ligand.

An improved method for synthesis and resolution of (R,S)-trans-7-hydroxy-2-[N-propyl-N-(3'-iodo-2'-propenyl)amino]tetralin (trans-7-OH-PlPAT,5), a new D3 dopamine receptor ligand, was reported. Both isomers, (R)-(+)- and (S)-(-)-5, were prepared and characterized. HPLC retention times obtained on a chiral column for these isomers were consistent with those observed for [125I]-(R)-(+)- and (S)-(-)-5. Direct radioiodination of an optically resolved tin precursor, (R)-(+)-7, yielded the desired [125I](R)-(+)-5, which is a simpler method for synthesis of this ligand. Binding studies with membrane preparations containing D3 dopamine receptors expressed in Spodoptera frugiperda (Sf9) cells also suggested that the [125I](R)-(+)-5 is the active isomer (Kd = 0.05 nM). The schemes described may provide an efficient way for synthesizing a large quantity of this new D3 dopamine receptor ligand for in vivo behavior studies.

Synthesis of (R,S)-trans-8-hydroxy-2-[N-n-propyl-N-(3'-iodo-2'-propenyl)amino]tetral in (trans-8-OH-PIPAT): a new 5-HT1A receptor ligand.

In order to develop tracers with higher specific activity to supplant the currently used [3H]-8-OH-DPAT [8-hydroxy-2-(N,N-di-n-propylamino)tetralin] for in vitro and in vivo evaluation of 5-HT1A receptors, a new radioiodinated ligand was prepared. (R,S)-trans-8- Hydroxy-2-[N-n-propyl-N-(3'-iodo-2'-propenyl)amino]tetralin (trans-8-OH-PIPAT), 8, was synthesized by a 10-step reaction. Binding studies with rat hippocampal membrane homogenates showed that 8 exhibited a Ki value of 0.92 nM against (R,S)-[3H]-8-OH-DPAT. Radiolabeled [125I]-8 was prepared from the corresponding tri-n-butyltin precursor via an oxidative iododestannylation reaction with sodium [125I]iodide. Binding studies in the hippocampal homogenates revealed that [125I]-8 bound to a single high-affinity site (Kd = 0.38 +/- 0.03 nM,Bmax = 310 +/- 20 fmol/mg of protein). Competition binding experiments clearly indicated that the new ligand displayed the expected 5-HT1A receptor binding profile. The rank order of potency was (R,S)-trans-8-OH-PIPAT > (R,S)- 8-OH-DPAT > WB4101 > 5-HT > (R,S)-trans-7-OH-PIPAT > (R,S)-7-OH-DPAT > (R,S)-propranolol > spiperone >> ketanserin >> dopamine > atropine. This new ligand offers several unique advantages, including high specific activity, high binding affinity, and low nonspecific binding, all of which make it an excellent probe for the investigation and characterization of 5-HT1A receptors.