scholarly journals Saturation Analysis in PET—Analysis of Errors Due to Imperfect Reference Regions

1994 ◽  
Vol 14 (2) ◽  
pp. 358-361 ◽  
Author(s):  
J.-E. Litton ◽  
H. Hall ◽  
S. Pauli
Keyword(s):  
Receptor Binding ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Position Emission Tomography ◽  
Reference Region ◽  
Nonspecific Binding ◽  
Binding Studies

In the determination of specific binding in receptor binding techniques in vitro as well as in vivo, determination of the nonspecific binding as well as the free component is of crucial importance. If a low proportion of specific binding is included when determining the nonspecific binding, relatively large errors may be obtained. In the present study, benzodiazepine (BZ) receptor binding in the human brain was determined in vivo using position emission tomography (PET) by applying a saturation procedure using [11C]flumazenil as an example of this problem. Analysis of the errors in Bmax and KD obtained using Scatchard analysis in PET was performed using a priori information from in vitro [3H]flumazenil binding in the pons, used normally as a reference region in BZ receptor binding studies. Even if the density of BZ receptors in the reference region pons is only 2% compared to that in the frontal cortex, this small proportion of specific binding sites will result in a 10% error in the Bmax and KD values. Simulation of a number of Scatchard plots was performed at varying ratios between the nonspecific and the specific binding.

Autoradiographic detection of kinin receptors in the human medulla of control, hypertensive, and diabetic donors

2002 ◽  
Vol 80 (4) ◽  
pp. 249-257 ◽  
Author(s):  
Hudson de Sousa Buck ◽  
Brice Ongali ◽  
Gaétan Thibault ◽  
Charles J Lindsey ◽  
Réjean Couture
Keyword(s):  
Receptor Binding ◽  
Binding Sites ◽  
Specific Binding ◽  
Inferior Olivary Nucleus ◽  
Binding Studies ◽  
Specific Binding Sites ◽  
Kinin Receptors ◽  
Medullary Nuclei ◽  
Inferior Olivary

Kinins have been elected to the status of central neuromediators. Their effects are mediated through the activation of two G-protein-coupled receptors, denoted B1 and B2. Functional and binding studies suggested that B1 and B2 receptors are upregulated in the medulla and spinal cord of hypertensive and diabetic rats. The aim of this study was to localize and quantify kinin receptors in post-mortem human medulla obtained from normotensive, hypertensive, and diabetic subjects, using in vitro receptor autoradiography with the radioligands [125I]HPP-HOE140 (B2 receptor) and [125I]HPP[des-Arg10]-HOE140 (B1 receptor). Data showed specific binding sites for B2 receptor (0.4–1.5 fmol/mg tissue) in 11 medullary nuclei from 4 control specimens (paratrigeminal > ambiguus > cuneate, gelatinous layer of the caudal spinal trigeminal nucleus > caudal and interpolar spinal trigeminal, external cuneate, solitary tract > hypoglossal > gracile > inferior olivary nuclei). Increased density of B2 receptor binding sites was observed in seven medullary nuclei of four hypertensive specimens (paratrigeminal > external cuneate > interpolar and caudal spinal trigeminal, gracile, inferior olivary > hypoglossal nuclei). B2 receptor binding sites were seemingly increased in the same medullary nuclei of two diabetic specimens. Specific binding sites for B1 receptor (1.05 and 1.36 fmol/mg tissue) were seen only in the inferior olivary nucleus in two out of the ten studied specimens. The present results support a putative role for kinins in the regulation of autonomic, nociceptive, and motor functions at the level of the human medulla. Evidence is also provided that B2 receptors are upregulated in medullary cardiovascular centers of subjects afflicted of cardiovascular diseases.Key words: bradykinin, hypertension, diabetes, human brain.

scholarly journals Fibrin and fibrinogen degradation products with an intact D-domain C- terminal gamma chain inhibit an early step in accessory cell-dependent lymphocyte mitogenesis

Blood ◽  
1993 ◽  
Vol 81 (11) ◽  
pp. 3006-3014 ◽  
Author(s):  
SC Robson ◽  
R Saunders ◽  
LR Purves ◽  
C de Jager ◽  
A Corrigall ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Degradation Products ◽  
Specific Binding ◽  
Lymphocyte Transformation ◽  
Mononuclear Leukocytes ◽  
Accessory Cell ◽  
Binding Studies ◽  
Gamma Chain

Abstract Although the low molecular weight degradation products of fibrinogen (FgDP) and fibrin (FbDP) are known to inhibit lymphocyte blastogenesis, the effect of purified macro-molecular FgDP and FbDP (molecular weight, 90 to 200 Kd) is unclear. We have examined the effect of these latter FgDP and FbDP and find that products that contain the D domain inhibit lymphocyte proliferation in response to T-cell mitogens, allogeneic mononuclear leukocytes, and anti-CD3 in vitro. Plasmic digestion of D1 in the absence of calcium with removal of the C-terminal end of the gamma chain or disruption of the gamma-gamma C-terminal cross-link site of D-dimer (DD) by puffadder venom (PAV-D) abrogates their inhibitory potential. Prior incubation of monocytes with DD or D1 inhibits subsequent lymphocyte transformation. Binding studies with radiolabeled DD and PAV-D confirm that monocytes interact only with DD. This specific binding may be competitively inhibited by monoclonal antibodies to CD11b/CD18 or by peptide analogues of the C-terminal gamma chain of fibrinogen that mimic the adhesion recognition site of integrins. We postulate that DD and D1 bind to CD11b/CD18 on adherent monocytes and modulate lymphocyte activation. These products are typically present in the plasma of patients with disseminated intravascular coagulation with sepsis and could therefore influence inflammatory processes in vivo.

scholarly journals A quantitative binding model for the Apl protein, the dual purpose recombination-directionality factor and lysis-lysogeny regulator of bacteriophage 186

2020 ◽  
Vol 48 (16) ◽  
pp. 8914-8926
Author(s):  
Erin E Cutts ◽  
J Barry Egan ◽  
Ian B Dodd ◽  
Keith E Shearwin
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Attachment Site ◽  
Binding Energies ◽  
Sequence Specificity ◽  
Binding Studies ◽  
Binding Model ◽  
Specific Binding Sites

Abstract The Apl protein of bacteriophage 186 functions both as an excisionase and as a transcriptional regulator; binding to the phage attachment site (att), and also between the major early phage promoters (pR-pL). Like other recombination directionality factors (RDFs), Apl binding sites are direct repeats spaced one DNA helix turn apart. Here, we use in vitro binding studies with purified Apl and pR-pL DNA to show that Apl binds to multiple sites with high cooperativity, bends the DNA and spreads from specific binding sites into adjacent non-specific DNA; features that are shared with other RDFs. By analysing Apl's repression of pR and pL, and the effect of operator mutants in vivo with a simple mathematical model, we were able to extract estimates of binding energies for single specific and non-specific sites and for Apl cooperativity, revealing that Apl monomers bind to DNA with low sequence specificity but with strong cooperativity between immediate neighbours. This model fit was then independently validated with in vitro data. The model we employed here is a simple but powerful tool that enabled better understanding of the balance between binding affinity and cooperativity required for RDF function. A modelling approach such as this is broadly applicable to other systems.

Characterization of the radioiodinated analogue of SCH 23390: In vitro and in vivo D-1 dopamine receptor binding studies

Life Sciences ◽  
1988 ◽  
Vol 43 (14) ◽  
pp. 1151-1160 ◽  
Author(s):  
Robert D. McQuade ◽  
Richard Chipkin ◽  
Nordine Amlaiky ◽  
Marc Caron ◽  
Louis Iorio ◽  
...  
Keyword(s):  
Dopamine Receptor ◽  
Receptor Binding ◽  
Sch 23390 ◽  
Binding Studies ◽  
Dopamine Receptor Binding

scholarly journals Fibrin and fibrinogen degradation products with an intact D-domain C- terminal gamma chain inhibit an early step in accessory cell-dependent lymphocyte mitogenesis

Blood ◽  
1993 ◽  
Vol 81 (11) ◽  
pp. 3006-3014
Author(s):  
SC Robson ◽  
R Saunders ◽  
LR Purves ◽  
C de Jager ◽  
A Corrigall ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Degradation Products ◽  
Specific Binding ◽  
Lymphocyte Transformation ◽  
Mononuclear Leukocytes ◽  
Accessory Cell ◽  
Binding Studies ◽  
Gamma Chain

Although the low molecular weight degradation products of fibrinogen (FgDP) and fibrin (FbDP) are known to inhibit lymphocyte blastogenesis, the effect of purified macro-molecular FgDP and FbDP (molecular weight, 90 to 200 Kd) is unclear. We have examined the effect of these latter FgDP and FbDP and find that products that contain the D domain inhibit lymphocyte proliferation in response to T-cell mitogens, allogeneic mononuclear leukocytes, and anti-CD3 in vitro. Plasmic digestion of D1 in the absence of calcium with removal of the C-terminal end of the gamma chain or disruption of the gamma-gamma C-terminal cross-link site of D-dimer (DD) by puffadder venom (PAV-D) abrogates their inhibitory potential. Prior incubation of monocytes with DD or D1 inhibits subsequent lymphocyte transformation. Binding studies with radiolabeled DD and PAV-D confirm that monocytes interact only with DD. This specific binding may be competitively inhibited by monoclonal antibodies to CD11b/CD18 or by peptide analogues of the C-terminal gamma chain of fibrinogen that mimic the adhesion recognition site of integrins. We postulate that DD and D1 bind to CD11b/CD18 on adherent monocytes and modulate lymphocyte activation. These products are typically present in the plasma of patients with disseminated intravascular coagulation with sepsis and could therefore influence inflammatory processes in vivo.

Age-related changes in [125I]hLH specific binding to rat interstitial cells

1981 ◽  
Vol 96 (4) ◽  
pp. 569-576 ◽  
Author(s):  
O. P. F. Clausen ◽  
K. Purvis ◽  
V. Hansson
Keyword(s):  
Specific Binding ◽  
Interstitial Cells ◽  
Scatchard Analysis ◽  
Age Related ◽  
Count Distribution ◽  
Rat Interstitial Cells ◽  
Binding Cell ◽  
Steroid Dehydrogenase

Abstract. Enriched Leydig cell suspensions were prepared from rats ranging from 5 to 80 days of age. The cells were incubated with [125I]hLH in vitro, and the proportion of LH binding cells determined by means of autoradiography. The proportion of 3β-hydroxy-steroid-dehydrogenase (3β-HSD) positive cells was also determined. By means of Scatchard analysis and determination of grain counts of LH positive cells, an increase in LH receptors per LH binding cells with increasing age was found. LH binding cells from animals 5 and 10 days of age had few LH receptors, more than a doubling occurred between day 10 and day 20, a significant increase was seen between day 20 and 40 whereas only a slight increase in LH receptors per LH binding cell was seen thereafter. Scatchard analysis and grain count analysis of labelled cells gave essentially the same results. The grain count distribution showed cells distributed around low grain values in animals of all ages, whereas a subpopulation of cells with high grain counts appeared in maturing and mature animals. Only a small fraction of LH positive cells had detectable levels of 3β-HSD activity in animals 5 and 10 days of age. With increasing age the proportion of 3β-HSD positive cells approached that of LH binding cells, indicating that rat interstitial cells aquire LH receptors well before any 3β-HSD activity can be traced.

Effects of heparin treatments in vivo and in vitro on adrenal angiotensin II receptors and angiotensin II-induced aldosterone production in rats

1988 ◽  
Vol 119 (3) ◽  
pp. 367-372 ◽  
Author(s):  
Sadahide Azukizawa ◽  
Ikuyo Iwasaki ◽  
Toshikazu Kigoshi ◽  
Kenzo Uchida ◽  
Shinpei Morimoto
Keyword(s):  
Angiotensin Ii ◽  
Benzyl Alcohol ◽  
Specific Binding ◽  
Angiotensin Ii Receptors ◽  
Scatchard Analysis ◽  
Aldosterone Production ◽  
Glomerulosa Cells ◽  
Heparin Preparation

Abstract. To evaluate the heparin effects in vivo and in vitro on adrenal angiotensin II receptors and angiotensin II-induced aldosterone production, we examined the angiotensin II binding and the maximum angiotensin II-induced aldosterone production using adrenal glomerulosa cells from rats treated with a heparin preparation containing benzyl alcohol (1500 IU/kg, twice daily for 6 weeks) or cells to which heparin (300 IU/l) was directly added. Comparison was made using the cells from rats treated with vehicle or the cells to which vehicle was directly added. Specific binding of [125I]iodo-angiotensin II was decreased in the cells from heparin-treated rats or in the heparin-treated cells. Scatchard analysis showed that the decrease in binding was due to a decrease in both the number and the affinity of angiotensin II receptors in the cells from heparin-treated rats and a decrease in the number, but not the affinity, of the receptors in the heparin-treated cells. Heparin also caused a decrease in the maximum angiotensin Il-induced production, but not the basal production, of aldosterone in the cells from heparin-treated rats and in the heparin-treated cells. These data suggest that heparin interacts with adrenal angiotensin II receptors to inhibit the angiotensin Il-induced aldosterone production.

scholarly journals Assessment of Extrastriatal Vesicular Monoamine Transporter Binding Site Density Using Stereoisomers of [11C]Dihydrotetrabenazine

1999 ◽  
Vol 19 (12) ◽  
pp. 1376-1384 ◽  
Author(s):  
Robert A. Koeppe ◽  
Kirk A. Frey ◽  
David E. Kuhl ◽  
Michael R. Kilbourn
Keyword(s):  
Binding Site ◽  
Specific Binding ◽  
Three Dimensional ◽  
Precise Determination ◽  
Tracer Uptake ◽  
Vesicular Monoamine Transporter ◽  
Reference Region ◽  
Monoamine Transporter

Previous studies have demonstrated the utility of [nC]dihydrotetrabenazine ([11C]DTBZ) as a ligand for in vivo imaging of the vesicular monoamine transporter system. The (+)-isomer has a high affinity (approximately 1 nmol/L) for the vesicular monoamine transporter (VMAT2) binding site, whereas the (–)-isomer has an extremely low affinity (approximately 2 μmol/L). Efforts to model dynamic (+)-[11C]DTBZ data demonstrate the difficulty in separating the specific binding component from the free plus nonspecific component of the total positron emission tomography (PET) measure. The authors' previous*** PET work, as well as in vitro studies, indicate that there is little specific VMAT2 binding in neocortical regions. However, precise determination of in vivo binding levels have not been made, leaving important questions unanswered. At one extreme, is there sufficient specific binding in cortex or other extrastriate regions to be estimated reliably with PET? At the other extreme, is there sufficiently little binding in cortex so that it can be used as a reference region representing nonsaturable tracer uptake? The authors address these questions using paired studies with both active (+) and inactive (–) stereoisomers of [11C]DTBZ. Six normal control subjects were scanned twice, 2 hours apart, after injections of 16 mCi of (+)- and (–)-[11C]DTBZ (order counter-balanced). Three-dimensional PET acquisition consisted of 15 frames over 60 minutes for each scan. Arterial samples were acquired throughout, plasma counted, and corrected for radiolabeled metabolites. Analysis of specific binding was assessed by comparison of total distribution volume measures from the (+)- and (–)-[11C]DTBZ scans. The authors' findings indicate that only approximately 5% of the cortical signal in (+)-[11C]DTBZ scans results from binding to VMAT2 sites. The strongest extrastriatal signal comes from the midbrain regions where approximately 30% of the PET measure results from specific binding. The authors conclude that (1) the density of VMAT2 binding sites in cortical regions is not high enough to be quantified reliably with DTBZ PET, and (2) binding does appear to be low enough so that cortex can be used as a free plus nonspecific reference region for striatum.

Metabolism and receptor binding of nandrolone and testosterone under in vitro and in vivo conditions

1985 ◽  
Vol 110 (3_Suppla) ◽  
pp. S31-S37 ◽  
Author(s):  
E. W. Bergink ◽  
J. A. A. Geelen ◽  
E. W. Turpijn
Keyword(s):  
Androgen Receptor ◽  
Receptor Binding ◽  
Reductase Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
In Vivo Studies ◽  
Muscular Tissue ◽  
Binding Studies ◽  
Tissue Homogenates

Abstract. The metabolism and receptor binding of nandrolone (N) and testosterone (T) were studied under in vitro and in vivo conditions. The results of both in vitro incubation studes with 3H-N and 3H-T in tissue homogenates from rats and in vivo infusion studies with 3H-N and 3H-T in conscious rats show the importance of the enzymes 5α-reductase and 3α/β-hydroxysteroid-oxidoreductases in the prostate and the importance of the enzyme 17β-hydroxysteroid dehydrogenase in the kidney for the effects of N and T on these tissues. Following infusion of a combined dose of 3H-N and 3H-T there is a preferential retention at the receptor of 5α-dihydrotestosterone (DHT) over 5α-dihydronandrolone (DHN), N and T (DHT ⪢ DHN > N > T) in the prostate because T is a better substrate than N for 5α-reductase and because DHT binds more strongly to the androgen receptor than DHN, N and T. In the kidney 5α-reductase is not important; there is a preferential retention of N in T (DHN and DHT were only present in small amounts) because N is less susceptible than T for metabolic inactivation by the enzyme 17β-hydroxysteroid dehydrogenase and N binds strongly to the androgen receptor. Both in vitro and in vivo studies show that N and T were relatively stable in spleen, thymus and muscular tissue (only shown in vivo) and, as a result, the same amount of N and T was bound to the receptor in these tissues in the in vivo infusion experiment. In vitro binding studies with the androgen receptor in intact human cells show that 5α-reduction increases the affinity of T and decreases the affinity of N and of the 17α-ethyl derivative of N (3-keto-ethylestrenol). The results of the present studies explain the relatively strong effect of N, or derivatives of N, compared to that of T on tissues devoid of 5α-reductase activity (e.g. muscular tissue) and they suggest that in particular there may be a strong effect of N on tissues which in addition have a high 17β-hydroxysteroid dehydrogenase activity (e.g. kidney).

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