Maximal Binding Affinity Research Articles

TraM protein of plasmid R1: In vitro selection of the target region reveals two consensus 7 bp binding motifs spaced by a 4 bp linker of defined sequence

The TraM protein encoded by plasmid R1 has three functions in conjugative transfer: (i) it positively controls transfer gene expression, (ii) it stimulates efficient site-specific ssDNA cleavage at the oriT in vivo and (iii) it couples the relaxosome to the envelope-bound transport complex. Plasmid R1 contains two binding regions for TraM, sbmA and sbmB, either of which comprises several minimal TraM targets. SELEX of a randomized minimal (18 bp) TraM binding sequence was used to search for sequences that bind TraM most efficiently. This approach resulted in a sequence with a modular structure with two 7 bp consensus motifs 5′ T 1G 2A 3N 4T 5C 6R 7 and 5′ T 1G 2A 3N 4T 5Y 6R 7 spaced by a 4 bp linker with the consensus sequence 5′ Y 1A 2/C 2T 3/G 3A 4. EMSAs revealed that evolution in vivo was for maximal binding affinity. R1 derivatives with mutated sbmA and sbmB sites based on one in vitro selected sequence were found to be either 5-fold impaired ( mut sbmA) in conjugation or could not be transferred by conjugation anymore ( mut sbmB). Transcriptional lacZ fusions demonstrated that the introduced sbmB mutations affected promoter activity severely and abrogated autoregulation by TraM, thereby explaining this drastic effect on conjugal transfer. The implications of these findings are discussed in the context of the role of TraM.

The N-terminal Arm of the Helicobacter pylori Ni2+-dependent Transcription Factor NikR Is Required for Specific DNA Binding

The Ni(2+)-dependent transcription factor NikR is widespread among microbes. The two experimentally characterized NikR orthologs, from Helicobacter pylori and Escherichia coli, display vastly different regulatory capabilities in response to increased intracellular Ni(2+). Here, we demonstrate that the nine-residue N-terminal arm present in H. pylori NikR plays a critical role in the expanded regulatory capabilities of this NikR family member. Specifically, the N-terminal arm is required to inhibit NikR binding to low affinity and nonspecific DNA sequences and is also linked to a cation requirement for NikR binding to the nixA promoter. Site-directed mutagenesis and arm-truncation variants of NikR indicate that two residues, Asp-7 and Asp-8, are linked to the cation requirement for binding. Pro-4 and Lys-6 are required for maximal DNA binding affinity of the full-length protein to both the nixA and ureA promoters. The N-terminal arm is highly variable among NikR family members, and these results suggest that it is an adaptable structural feature that can tune the regulatory capabilities of NikR to the nickel physiology of the microbe in which it is found.

Hepatic sequestration of chlordecone and hexafluoroacetone evaluated by pharmacokinetic modeling

Chlordecone (CD) and mirex (M) differ by a single carbonyl group in CD in place of two chlorines in M. Although both compounds are lipophilic, their tissue distributions differ markedly: CD concentrations are highest in liver; M concentrations are highest in fat. We used tissue time course data in rats from our laboratory for CD and M and literature data from monkeys to develop PBPK models to study differences in liver and fat partitioning. The PK model for M had partitioning in tissue without specific hepatic binding. The CD model had partitioning similar to M, and also included liver binding: the maximal binding ( B max) and binding affinity constant ( Kd) required to describe the rat data were 370 nmol/g liver and 100 nM, respectively. To see if other ketones with electron withdrawing constituents at the alpha carbon were also preferentially distributed to liver, we developed a PBPK description for tissue distribution of hexafluoroacetone (HFA). Compared to acetone, HFA is known to be preferentially sequestered in liver and more slowly excreted unchanged from the body. Acetone is more equally distributed to tissues. HFA distribution was evaluated with a PBPK model that included hepatic binding. B max and Kd were 1.58 μmol/g liver and 301 μM. In summary, liver sequestration of CD and HFA most likely represents relatively high-affinity but reversible binding of activated carbonyls in these compounds (activated by the presence of electron withdrawing substituents on the alpha-carbons) with glutathione and glutathione transferases, that are present at much higher concentrations in liver than in other tissues. Strong, but reversible hemithioketal formation with active sulfhydryls may also be associated with the toxic responses to CD and HFA.

Conformationally Constrained CCK8 Analogues Obtained from a Rationally Designed Peptide Library as Ligands for Cholecystokinin Type B Receptor

A library of 14 cyclic peptide analogues derived from the octapeptide C-terminal sequence of the human cholecystokinin hormone (CCK(26-33), or CCK8) was designed, synthesized, and characterized. The 14 peptide analogues were rationally designed to specifically interact with the CCK type B receptor (CCK(B)-R) on the basis of the structure of the bimolecular complex between CCK8 and the third extracellular loop of CCK(B)-R, namely CCK(B)-R(352-379). The rational design of new ligands for CCK(B)-R has relied on stabilization by cyclic constraints of the structural motifs that bring the key residues of the ligand (especially Trp 30, Met 31, and Phe 33) in the proper spatial orientation for optimal interaction with the receptor. The binding affinity of the new ligands for CCK(B)-R was assessed by displacement experiments of (111)In-radiolabeled CCK8 in cells that overexpress the CCK(B) receptor. The new ligands generally showed binding affinities lower than that of parent CCK8, with the best compounds having IC50 values around 10 microM. Structure-activity relationship data show that preservation of the Trp 30-Met 31 motif is essential and that the Phe 33 side chain must be present. NMR conformational studies of the compound with maximal binding affinity (cyclo-B11, IC50=11 microM) in DPC micelles shows that this compound presents a turn-like conformation centered at the Trp 30-Met 31 segment, as planned by rational design. Such a conformation is stabilized by its interaction with the micelle rather than by the cyclic constraint.

Chronic corticosterone treatment increases the endocannabinoid 2-arachidonylglycerol in the rat amygdala

This research was designed to examine the effect of three weeks of administration of corticosterone (20 mg/kg) on endocannabinoid content and cannabinoid CB 1 receptor binding in the amygdala. It was found that the endocannabinoid 2-arachidonylglycerol was significantly increased in the amygdala following chronic corticosterone treatment. However, there was no change in either the maximal binding ( B max) or binding affinity ( K D) of [ 3H]-CP 55,940 to the CB 1 receptor in the amygdala. Given the role of amygdalar endocannabinoids in the regulation of emotionality, this suggests that the ability of glucocorticoids to influence affective behavior may involve interactions with regulation of endocannabinoid content.

Transcriptional Repressor CopR: use of SELEX to study the copR operator indicates that evolution was directed at maximal binding affinity.

CopR is one of the two copy number control elements of the streptococcal plasmid pIP501. It represses transcription of the repR mRNA encoding the essential replication initiator protein about 10- to 20-fold by binding to its operator region upstream of the repR promoter pII. CopR binds at two consecutive sites in the major groove of the DNA that share the consensus motif 5'-CGTG. Previously, the minimal operator was narrowed down to 17 bp, and equilibrium dissociation constants for DNA binding and dimerization were determined to be 0.4 nM and 1.4 microM, respectively. In this work, we used a SELEX procedure to study copR operator sequences of different lengths in combination with electrophoretic mobility shift assays of mutated copR operators as well as copy number determinations to assess the sequence requirements for CopR binding. The results suggest that in vivo evolution was directed at maximal binding affinity. Three simultaneous nucleotide exchanges outside the bases directly contacted by CopR only slightly affected CopR binding in vitro or copy numbers in vivo. Furthermore, the optimal spacer sequence was found to comprise 7 bp, to be AT rich, and to need an A/T and a T at the 3' positions, whereas broad variations in the sequences flanking the minimal 17-bp operator were well tolerated.

Thermodynamics of DNA Binding and Distortion by the Hyperthermophile Chromatin Protein Sac7d

Sac7d is a hyperthermophile chromatin protein which binds non-specifically to the minor groove of duplex DNA and induces a sharp kink of 66 degrees with intercalation of valine and methionine side-chains. We have utilized the thermal stability of Sac7d and the lack of sequence specificity to define the thermodynamics of DNA binding over a wide temperature range. The binding affinity for poly(dGdC) was moderate at 25 degrees C (Ka = 3.5(+/-1.6) x 10(6) M(-1)) and increased by nearly an order of magnitude from 10 degrees C to 80 degrees C. The enthalpy of binding was unfavorable at 25 degrees C, and decreased linearly from 5 degrees C to 60 degrees C. A positive binding heat at 25 degrees C is attributed in part to the energy of distorting DNA, and ensures that the temperature of maximal binding affinity (75.1+/-5.6 degrees C) is near the growth temperature of Sulfolobus acidocaldarius. Truncation of the two intercalating residues to alanine led to a decreased ability to bend and unwind DNA at 25 degrees C with a small decrease in binding affinity. The energy gained from intercalation is slightly greater than the free energy penalty of bending duplex DNA. Surprisingly, reduced distortion from the double alanine substitution did not lead to a significant decrease in the heat of binding at 25 degrees C. In addition, an anomalous positive DeltaCp of binding was observed for the double alanine mutant protein which could not be explained by the change in polar and apolar accessible surface areas. Both the larger than expected binding enthalpy and the positive heat capacity can be explained by a temperature dependent structural transition in the protein-DNA complex with a Tm of 15-20 degrees C and a DeltaH of 15 kcal/mol. Data are discussed which indicate that the endothermic transition in the complex is consistent with DNA distortion.

Changes of mu and kappa opioid receptors in cathartic colon of rats

Objective: To observe the changes of mu and kappa opioid receptors in the cathartic colon of rat, and to clarify that whether opioid receptors accounts for the occurrence of slow transit constipation (STC). Methods: The cathartic colon model of rat was made by feeding with laxatives. The activity of mu and kappa opioid receptors in the cathartic colon of rat was measured by radio-ligand binding assay. Results: Compared with the control group, the maximal binding capacity (Bmax) and affinity(Kd) of mu opioid receptor in cathartic colon group were significantly increased (207.00±22.90 fmol/mg·p vs 82.00±14.23 fmol/mg·p, P 0.01;3.30±0.45 mmol/L vs 2.40±0.57 mmol/L,P 0.05). The maximal binding capacity of kappa opioid receptor also showed a great increase (957.00±102.41 fmol/mg·p vs 459.00±52.41 fmol/mg·p, P0.01), but no significant difference of affinity was found between the two groups. Conclusion: The mu and kappa opioid receptors may be involved in the functional disorders of cathartic colon.

Kinetics and Structural Requirements for the Binding Protein of the Di-tripeptide Transport System of Lactococcus lactis

The gene (dppA) encoding the binding protein of the di-tripeptide ABC transporter of Lactococcus lactis (DppA) was cloned under the control of the nisin promoter. Amplified expression ( approximately 200-fold increase) of the protein fused to a carboxyl-terminal six-histidine tag allowed the purification of DppA-(His)(6) by nickel-chelate affinity and anion-exchange chromatography. Ligand binding to DppA-(His)(6) elicited an electrophoretic mobility shift, a decrease in the intrinsic fluorescence, and a blue shift of the emission maximum. Each of these parameters detected conformational changes in the protein that reflect ligand binding, and these were used to determine the structural requirements of DppA-(His)(6) for binding peptides. The major features of peptide binding include (i) high affinity for di- and tripeptides, (ii) requirement of a free N-terminal alpha-amino group and an alpha-peptide bound contiguous with the N-terminal amino group, (iii) stereospecificity for L-isomers, and (iv) preference for dipeptides containing methionine or arginine, followed by hydrophobic tripeptides consisting of leucine or valine residues. Maximal binding affinity was detected at pH 6.0, and the K(d) for binding increased 1 order of magnitude for every unit increase in pH. This suggests that the ionization of protein residues (pK > 6.0) in or in close proximity to the binding site is critical in the binding mechanism.

Clomiphene Analogs with Activity In Vitro and In Vivo against Human Breast Cancer Cells

Six hundred triphenylethylenes were assayed for antiproliferative activity against MCF-7, LY2, and MDA-MB-231 breast cancer cells using sulforhodamine B dye to measure proliferation. Here we report on just 63 of the compounds, mostly clomiphene analogs, with substitutions on the α′ or β ring, at the vinyl position or in the side chain, of which 23 were active, as defined by antiproliferation ic 50 values ≤1 μM. Activity profiles showed that 23 and 11 analogs were active toward MCF-7 and LY2, respectively, but none were active against MDA-MB-231. The ic 50 values of tamoxifen were 2.0 μM against MCF-7 and 7.5 μM against LY2 and MDA-MB-231. Estradiol reversed antiproliferative activities of several E isomers but not their Z isomer counterparts. Clomiphene side chain analogs 46 [( E)-1-butanamine, 4-[4-(2-chloro-1,2-diphenylethenyl) phenoxy]- N, N-diethyl-dihydrogen citrate (MDL 103,323)] and 57 [( E)- N-[ p-(2-chloro-1,2-diphenylvinyl) phenyl]- N, N-diethylethylenediamine dihydrogen citrate (MDL 101,986)] were 4- to 5-fold more effective than tamoxifen. Methylene additions up to (CH 2) 12 in the clomiphene side chain showed that analog 46 [(CH 2) 4 side chain] had maximal antiproliferative activity, binding affinity, and inhibition of transcription of an estrogen response element luciferase construct in transfected MCF-7 cells. Intraperitoneal administration of 46 or 57 inhibited progression of MCF-7 breast tumor xenografts in nude mice with ed 50 values of <0.02 mg/mouse/day. Both analogs may hold promise for treating ER positive breast cancer and are of interest for further development.

Permeability properties of the porin of spinach leaf peroxisomes.

The membrane of spinach leaf peroxisomes contains an anion-selective channel. Reconstitution experiments were performed with lipid bilayer membranes to study its permeability properties. A variety of different monovalent inorganic and organic anions were found to be permeable through the porin channel. Its single-channel conductance for these different ions suggested that the channel has a minimum diameter of about 0.6 nm. From selectivity measurement in KCl solution a ratio of the anion permeability to cation permeability of less than 0.04 was determined, indicating an almost ideal selectivity of the peroxisomal channel for chloride. The permeation of chloride through the peroxisomal channel could be blocked efficiently by the addition of increasing concentrations of organic anions to the aqueous phase. The results are consistent with a binding site for dicarboxylic anions inside the peroxisomal channel. A particular high stability constant for the binding was obtained for peroxisomal metabolites such as malate, oxaloacetate, succinate, and 2-oxoglutarate, which have to cross the membrane of plant peroxisomes in vivo. Among these solutes maximal binding affinity was determined for C4 dicarboxylic anions. The results indicate that the peroxisomal channel does not form a general diffusion pore similarly to known eukaryotic porins, but has specific properties comparable to specific and inducible porins, which have been characterized in some gram-negative bacteria.

Resistance to the Antitumor Agent Gallium Nitrate in Human Leukemic Cells Is Associated with Decreased Gallium/Iron Uptake, Increased Activity of Iron Regulatory Protein-1, and Decreased Ferritin Production

The mechanism of drug resistance to gallium nitrate is not known. Since gallium can be incorporated into ferritin, an iron storage protein that protects cells from iron toxicity, we investigated whether ferritin expression was altered in gallium-resistant (R) CCRF-CEM cells. We found that the ferritin content of R cells was decreased, while heavy chain ferritin mRNA levels and iron regulatory protein-1 (IRP-1) RNA binding activity were increased. IRP-1 protein levels were similar in gallium-sensitive (S) and R cells, indicating that R cells contain a greater proportion of IRP-1 in a high affinity mRNA binding state. 59Fe uptake and transferrin receptor expression were decreased in R cells. In both S and R cells, gallium inhibited cellular 59Fe uptake, increased ferritin mRNA and protein, and decreased IRP-1 binding activity. Gallium uptake by R cells was markedly diminished; however, the sensitivity of R cells to gallium could be restored by increasing their uptake of gallium with excess transferrin. Our results suggest that R cells have developed resistance to gallium by down-regulating their uptake of gallium. In parallel, iron uptake by R cells is also decreased, leading to changes in iron homeostasis. Furthermore, since gallium has divergent effects on iron uptake and ferritin synthesis, its action may also include a direct effect on ferritin mRNA induction and IRP-1 activity.

Burn injury alters beta-adrenergic receptor and second messenger function in rat ventricular muscle

The molecular pharmacologic bases for the attenuated cardiovascular and metabolic responses to catecholamines, after burn injury, have not been elucidated. In the present study, myocardial tissues were used as a model of beta-adrenergic receptors to study burn injury-induced alterations in receptors and in signal transduction. Prospective study, randomized to treatment and control groups. University-hospital research laboratory. Male Sprague-Dawley rats (180 to 210 g). A 50% body surface area burn or sham-burn was administered to the rats. Myocardial membranes were isolated at 24 hrs, 7 days and 14 days after 50% body surface area scald or sham injury. (-)125I-iodocyanopindolol was used to assess maximal binding capacity and affinity of the beta-adrenergic receptor. Basal and stimulated concentrations of second messenger, cyclic adenosine monophosphate (cAMP), were also assessed. Production of cAMP during isoproterenol stimulation tested the integrity of the beta-adrenergic receptor-mediated signal transduction. Forskolin, which stimulates adenylate cyclase enzyme directly (bypassing the receptor and G protein) to produce cAMP, tested the efficacy of the enzyme itself. Maximal binding capacity was unaltered between the experimental and control groups, but the affinity (mean +/- SEM) was significantly decreased in burned animals at 7 days (125.4 +/- 15.5 picomoles [pmol]; p = .01) and at 14 days (216.7 +/- 50.7 pmol; p = .001) compared with controls (75.5 +/- 8.4 pmol). In different set experimental and control groups, basal concentrations of cAMP in myocardial membranes were significantly decreased in burned animals at 7 days (control 38.6 +/- 4.2 vs. 5.8 +/- 0.9 pmol/mg of protein/min; p = .003) and at 14 days (control 47.4 +/- 3.2 vs 28.3 +/- 6.6 pmol/mg of protein/min; p = .002). The forskolin (direct)-stimulated synthesis of cAMP was decreased in burned animals at 24 hrs (control 339.0 +/- 40.5 vs. 214.4 +/- 16.6 pmol/mg of protein/min; p = .01), at 7 days (control 289.0 +/- 34.4 vs. 32 +/- 13.0 pmol/mg of protein/min; p = .01), and at 14 days (control 322.9 +/- 28.6 vs. 137.0 +/- 46.1 pmol/mg of protein/min; p = .01). The isoproterenol or receptor-mediated stimulation of cAMP production was also significantly (p < .001) impaired in burned animals compared with controls at 24 hrs (control 134.7 +/- 11.9 vs. 83.1 +/- 13.3 pmol/mg of protein/min), and at 14 days (control 128.2 +/- 7.2 vs. 92.8 +/- 17.7 pmol/mg of protein/min). The etiology of the decreased responses in the myocardium to exogenous and endogenous beta-adrenergic receptor agonists after burn injury may be attributed to decreased affinity for ligands, and also to impaired receptor-mediated signal transduction and to decreased adenylate cyclase enzyme activity, resulting in decreased basal and stimulated second messenger (cAMP) production.

TUMOR NECROSIS FACTOR-α ADMINISTRATION INCREASES KUPFFER CELL CYCLIC ADENOSINE MONOPHOSPHATE LEVELS

Although studies have indicated that both Kupffer cell cyclic adenosine monophosphate (cAMP) and circulating TNF levels increase following trauma-hemorrhage and resuscitation, it remains unknown whether the elevated TNF levels are responsible for the increased Kupffer cell cAMP levels. To determine this, recombinant murine TNF-alpha (1.2 x 10(7) U/mg) was infused intravenously (.25 mg/kg body wt) over 30 min in normal rats. At 1 h after TNF-alpha or vehicle infusion, Kupffer cells and hepatocytes were isolated and cAMP levels were determined by radioimmunoassay. The levels of cAMP in the spleen and kidney were also measured. In addition, the maximal binding capacity and affinity of beta-adrenergic receptors were determined in Kupffer cells and hepatocytes by using [125I]iodopindolol. To determine whether there is any correlation between Kupffer cell cAMP and prostaglandin E2 (PGE2) or epinephrine, plasma levels of catecholamines and PGE2 were measured. The results indicated that TNF-alpha infusion significantly increased Kupffer cell cAMP levels while hepatocyte cAMP levels were not altered. Moreover, cAMP levels also increased in the macrophage/lymphocyte-rich spleen but were not altered in the kidney. Kupffer cell beta-receptor binding characteristics were not significantly affected by TNF-alpha infusion. In contrast, TNF-alpha administration markedly increased plasma levels of PGE2 and epinephrine. Thus, the elevated Kupffer cell cAMP levels induced by TNF-alpha are not due to upregulation of beta-adrenergic receptors, but may be associated with the elevated levels of circulating PGE2 and/or epinephrine.

Effects of age and endurance training on β-adrenergic receptor characteristics in Fischer 344 rats

The purpose of this investigation was to examine changes in β-adrenergic receptor characteristics in various tissues with age and endurance training. Forty-eight young (6 months), middle-aged (15 months), and old (25 months) male Fischer 344 rats were assigned to either a trained or sedentary running group. Animals were endurance trained by 10 weeks of treadmill running at 75% maximal capacity, 1 h/day, 5 days/week. Animals were sacrificed at rest and the heart, liver, and soleus were removed for analysis. Percent of high and low affinity binding sites were determined by competitive binding experiments. Competition curves were generated using 12 concentrations of ICI-89406 ( β 1 antagonist) and ICI-118551 ( β 2 antagonist) to inhibit the total binding of (−) [ 125I] pindolol (IPIN). Maximal binding site number ( B max) and affinity (K d ) were determined by Scatchard analysis. Heart B max did not differ with age or training. An aging effect was observed in liver such that middle-aged and old animals had greater B max compared to young animals. In soleus, B max was not altered with training but decreased with age. While training had no affect on affinity in the liver and soleus, heart affinity increased with training in both the middle-aged (21%) and old (27%) animals. In soleus, affinity increased but remained unaltered in heart and liver with age. The ratio of β 1: β 2 receptors in the heart and liver did not differ with age or training. The influence of age and training on β-adrenergic receptor characteristics appear to be tissue specific.

Molecular cloning and expression of a hexameric Drosophila heat shock factor subject to negative regulation

We report the cloning of the transcriptional activator of heat shock genes, HSF, from Drosophila. The predicted sequence.of Drosophila HSF protein is surprisingly divergent from that of yeast HSF, except in regions important for DNA binding and oligomerization. A segment of the DNA binding domain of HSF bears an intriguing similarity to the putative DNA recognition helix of bacterial sigma factors, while the oligomerization domain contains an unusual arrangement of conserved hydrophobic heptad repeats. Drosophila HSF produced in E. coli under nonshock conditions forms a hexamer that binds specifically to DNA with high affinity and activates transcription from a heat shock promoter in vitro. In contrast, when HSF is expressed in Xenopus oocytes, maximal DNA binding affinity is observed only after heat shock induction. These results suggest that Drosophila HSF has an intrinsic affinity for DNA, which is repressed under nonshock conditions in vivo.

125I]-Omega conotoxin binding to human frontal cortex from normal, Alzheimer's and non-Alzheimer's dementia patients

We studied the binding of the calcium antagonist neurotoxin [ 125I]-omega conotoxin (GVIA) in age-matched human brains from normal, Alzheimer's disease and non-Alzheimer's dementia patients. Crude preparations of plasmalemmal membranes from frontal cortex were utilized. Saturation isotherms were subjected to Scatchard analysis to determine maximal binding capacity (B max) and binding affinity (K d). In all brain samples tested, [ 125I]-GVIA binding was homogenous to a single class of high affinity binding sites. Scatchard analysis of saturation isotherms gave the following estimates for normal brains (mean ± S.D., n = 7): B max = .630 ± .200 pmol/mg and K d = .177 ± .054 nM. No significant change was observed in the K d or B max estimates for [ 125I]-omega conotoxin binding in Alzheimer's disease or non-Alzheimer's dementia brains when compared to normal brains. Although these findings do not rule out the existence of localized changes in calcium channel receptor binding in the frontal cortex of Alzheimer's disease patients, the results do suggest that the neuronal voltage sensitive calcium channel may be unaltered in Alzheimer's disease.

Neuropeptide-dopamine interactions. IV. Effect of thyrotropin-releasing hormone on striatal dopaminergic neurons

Many biologic effects of TRH seem to be mediated via a dopaminergic mechanism. The present study examined the effects of chronic TRH administration on the properties of nigrostriatal dopaminergic neurons. Ten days, continuous subcutaneous TRH administration via an osmotic minipump led to a significant rise in striatal level of 3,4-dihydroxyphenylacetic acid, but not of homovanillic acid or dopamine. These treatments also did not elicit any significant changes in the maximal binding capacity (B max) or affinity (K D) of either D 1- or D 2-dopamine receptor. By contrast, TRH administration led to a significant increase in both B max and K D of striatal mazindol binding. This effect of TRH, however, was not observed in in vitro studies. In conclusion, these data suggest that in vivo administration of TRH may modulate dopaminergic activities by altering, directly or indirectly, dopamine release and reuptake.

Androgen insensitivity in oligospermic men: a reappraisal.

Androgen insensitivity has been reported to be present in as many as 40% of patients with severe oligospermia. In order to evaluate further the role of androgen resistance in male infertility we studied 24 men with severe oligospermia. Plasma T and LH were measured by RIA and the T X LH product was calculated. Fibroblasts were grown from genital skin obtained during testicular biopsies and androgen receptor maximal binding capacity (BMAX) and affinity (KD) were measured in fibroblast monolayers. Pubic skin 5 alpha-reductase activity, an androgen-dependent enzyme, was measured in skin homogenates. Plasma T values were in the upper normal range [7.0 +/- 1.7 (SEM) ng ml-1] whereas the T X LH product was high (greater than 50) in only six patients. Mean BMAX and KD values for the androgen receptor were normal [BMAX: 788 +/- 259 fmol mg DNA-1 (patients, n = 20), 726 +/- 227 (normal men, n = 20), and KD: 0.27 +/- 0.24 (patients, n = 20), 0.18 +/- 0.09 (normal men, n = 15), respectively]. However, four men had supranormal KD values. The mean BMAX was also normal when the group of men with sperm densities below 10(6) per ejaculate was considered separately. Public skin 5 alpha-reductase activity was normal in all but four patients (patients: 177.1 +/- 91 fmol/mg skin/h, n = 30, normal men: 210 +/- 45, n = 20 patients). In conclusion, androgen receptor BMAX levels were normal in all patients studied, regardless of the sperm density and the T X LH product. Pubic skin 5 alpha-reductase activity was also normal in all but four patients. In these four patients, a qualitative defect of the androgen receptor cannot be excluded. In this group of patients with severe oligospermia, infertility did not seem to be related to quantitative abnormality of the androgen receptor as was previously reported.

Effect of pyridoxine deficiency in young rats on high-affinity serotonin and dopamine receptors.

The high-affinity bindings of [3H]-5-hydroxytryptamine to serotonin S-1 receptors, [3H]-ketanserin to serotonin S-2 receptors in the cerebral cortex, [3H]-fluphenazine to dopamine D-1 receptors, and [3H]-spiroperidol to dopamine D-2 receptors in the corpus striatum were studied in pyridoxine-deficient rats and compared to pyridoxine-supplemented controls. There was a significant increase in the maximal binding (Bmax) of serotonin S-1 and S-2 receptors with a significant decrease in their binding affinities (Kd). However, there were no significant changes either in the maximal binding or binding affinity of striatal dopamine D-1 and D-2 receptors. Receptor sensitivity seems to correlate negatively with the corresponding neurotransmitter concentrations in the pyridoxine-deficient rats.