The titration of tetanus antitoxin I. Factors affecting the sensitivity of the indirect haemagglutination test

Various factors affecting the indirect HA test for the titration of tetanus antitoxin have been evaluated with a view to obtaining maximum sensitivity in tests using unfixed sheep erythrocytes and sheep erythrocytes fixed with glutaraldehyde, formaldehyde and pyruvic aldehyde. The optimal concentration of tannic acid has been found to be 1/40 000 for tanning both fixed and unfixed sheep erythrocytes. Tanned sheep erythrocytes sensitized with 50 Lf/ml of tetanus toxoid at pH 7.2 for one hour were the most sensitive. Although the optimal temperature of sensitization was found to be 56 degrees C, unfixed cells tended to clump and lyse at this temperature. Thus a temperature of 37 degrees C was used to sensitize unfixed sheep erythrocytes. Sheep erythrocytes from different animals and the final concentration of sensitized sheep erythrocytes both had great effects on sensitivity. A final concentration of 0.5% of sensitized sheep erythrocytes was found suitable as a compromise between sensitivity and readability. The loss of sensitivity of fixed and sensitized erythrocytes was investigated by storing these cells at 4-8 degrees C for six to nine months.     

Cytochemical studies on the localization of methanol oxidase and other oxidases in peroxisomes of methanol-grown Hansenula polymorpha

The localization of methanol oxidase activity in cells of methanol-limited chemostat cultures of the yeast Hansenula polymorpha has been studied with different cytochemical staining techniques. The methods were based on enzymatic or chemical trapping of the hydrogen peroxide produced by the enzyme during aerobic incubations of whole cells in methanol-containing media. The results showed that methanol-dependent hydrogen peroxide production in either fixed or unfixed cells exclusively occurred in peroxisomes, which characteristically develop during growth of this yeast on methanol. Apart from methanol oxidase and catalase, the typical peroxisomal enzymes d-aminoacid oxidase and l--hydroxyacid oxidase were also found to be located in the peroxisomes. Urate oxidase was not detected in these organelles. Phase-contrast microscopy of living cells revealed the occurrence of peroxisomes which were cubic of form. This unusual shape was also observed in thin sections examined by electron microscopy. The contents of the peroxisomes showed, after various fixation procedures, a completely crystalline or striated substructure. It is suggested that this substructure might represent the in vivo organization structure of the peroxisomal enzymes.     

Effects of phase transitions on enzymes bound to different cellular membranes

Summary In unfixed cryostat sections enzymes attached to various cellular membranes differ in their sensitivity to the inhibitory effects of Ca and CNS ions. Similar differences between enzymes related to various membranes were also noted in the possibility of reversing the inhibition of NaCl treatment and in the effects of oxygen in rendering the inhibition irreversible.Evidence was adduced to show that some of the previous data on reversibility of ionic inhibition of membrane bound enzymes were due to an inadvertently introduced step of drying. The effect of drying was not apparent in NaCl-treated sections. In CaCl²- and KCNS-treated sections the effect of drying was due to a high concentration of the salts prevailing during evaporation and to atmospheric oxygen. A possible effect of denaturation not associated with oxidation could not be excluded.A summary of this report has been presented at the International Congress of Histochemistry and Cytochemistry in New York in August 1968.     

Ultrastructural localization of elastase-like enzymes in human neutrophils

The thiol ester, N-t-butyloxycarbonyl-L-alanine-p-nitrothiophenyl ester (Boc-Ala-SNp) has been synthesized and applied as an ultrastructural cytochemical substrate for leukocytic elastase-like enzymes. Incubation of fixed human neutrophils with Boc-Ala-SNp in the presence of gold ions generates electron-dense deposits of gold p-nitrothiophenolate in the nuclear membrane, endoplasmic reticulum, Golgi complex, mitochondria, and granules. Deposition of product is inhibited by pretreatment of cells with general and specific chemical inactivators of neutrophil elastase. This substrate appears to have significant potential as a probe for the ultrastructural localization of elastase-like activity.     

Unique cathepsin D-type proteases in rat thoracic duct lymphocytes and in rat lymphoid tissues.

Two unique cathepsin D-type proteases apparently present only in rat thoracic duct lymphocytes and in rat lymphoid tissues are described. One, termed H enzyme, has an apparent molecular weight of similar to95,000; the other, termed L enzyme, has an apparent molecular weight of similar to45,000, in common with that of most cathepsins D from other tissues and species. Both enzymes differ from cathepsin D, however, by a considerably greater sensitivity to inhibition by pepstatin and by a smaller degree of inhibition by an antiserum which inhibits rat liver cathepsin D. H enzyme is converted to L enzyme by treatment with beta-mercaptoethanol; the relationship between the two enzymes remains unknown. H and L enzyme have been detected in rat lymphoid tissues and in mouse spleen, but they are not present in other rat tissues (liver, kidney, adrenals), rabbit tissues, calf thymus, bovine spleen, or human tonsils. As measured on acid-denatured bovine hemoglobin as substrate, both enzymes have pH activity curves identical with that of rat liver cathepsin D, with optimal activity at pH 3.6. Activity on human serum albumin is much less and also shows an optimum at pH 3.6; hence, neither enzyme has the properties of cathepsin E. Thiol-reactive inhibitiors have no effect on the activity of H and L enzyme; thus they do not belong to the B group of cathepsins. Additional information, discussed in this paper, leads us to conclude that partially purified H and L enzymes are cathepsin D-type proteases.     

Alkaline phosphatases in fixed plant cells

Alkaline phosphatase activity in fixed plant cells has now been demonstrated cytochemically. Presumably cytochemical findings on plant alkaline phosphatases had been lacking because glycerophosphate, which is not hydrolyzed by fixed plant cells, had been used as the substrate.Alkaline phosphatase activity in the onion and corn nuclei has been compared with the activity in rat tissues. In the plant tissues, hydrolysis of phosphates was demonstrated when the substrates guanylic acid, adenosine diphosphate, adenosine triphosphoric acid, diphosphopyridine nucleotide, hexosediphosphates and inorganic pyrophosphate and metaphosphate were used. When the substrates glycerophosphate, adenylic acid and hexosemonophosphates were used, hydrolysis was not found. In the animal tissues however, hydrolysis was demonstrated of all organic phosphoesters employed and of sodium metaphosphate but not the hydrolysis of sodium pyrophosphate.One alkaline phosphatase found in the fixed plant tissues specifically hydrolyzed guanylic acid but no other nucleotide and one specifically hydrolyzed metaphosphate to orthophosphate.The enzymes in both plant and animal cells which hydrolyzed metaphosphates and pyrophosphates were found to require magnesium ions for their activity and to be inhibited by fluoride ions.“Alkaline, phosphatase,” so intimately associated with the chromatin in the nucleus, is postulated to be not just one enzyme but a number of enzymes.     

Cytochemistry and biochemistry of acid phosphatases. III. Inhibition experiments of lysosomal and secretory acid phosphatases of the rat ventral prostate

Biochemical and cytochemical inhibition experiments of rat prostatic acid phosphatase were performed using enzymes separated on isoelectric focusing (IEF) gels, and thin sections of the rat ventral prostate. Various inhibitors, including L (+) tartrate, mercuric ions and sodium fluoride were applied to electrofocused enzymes which were subsequently stained for acid phosphatase activity. Enzymes focused on IEF gels at pH 7.9 and 8.1, respectively, were inhibited with 1.8 x 10-3 M tartrate, while the enzyme activities with isoelectric points (pl) of 5.6 and 7.15, respectively, were only slightly inhibited by this compound. Using 10-3M mercuric ions, enzymes with pl of 5.6 and 7.15 were inhibited while the enzymes with pl of 7.9 and 8.1 were still active. The biochemical procedures were adapted to chopper sections of perfused-fixed ventral prostate of the rat. Preincubation of the sections with 2.4 x 10-3M mercuric chloride blocked the secretory enzyme and most of the lysosomal enzyme and resulted in an artificial staining of the Golgi apparatus and other cytoplasmic organelles. Nuclear precipitates however were prevented. L (+) tartrate could not be used at the ultrastructural level since it developed false positive results by the formation of lead tartrate. The results indicate that no selective inhibition of either secretory or lysosomal acid phosphatase can be achieved at the ultrastructural level using metal salts or tartrate, respectively.     

Stable acid phosphatase: II. Effects of pH and inhibitors

Microdensitometry demonstrated that stable acid phosphatase (SAPhase) in rat and hamster osteoclasts, chondroclasts, and chondrocytes has very similar properties. The differences that were observed suggest that conformational alterations in the enzymes may be responsible for inhibition by some agents such as tartrate. These differences in response to inhibitors depend on the method of embedding as well as on species differences. SAPhase appears to correspond to acid nitrophenyl posphatase, as shown by its pH dependent re-activation, resistance to fluoride inhibition at near-neutral pH, and the inverse effect of pH on inhibition by zinc versus aluminium ions. That proportion of SAPhase resistant to fluoride is an acid phosphatase with activity at near-neutral pH rather than a strict neutral phosphatase. The difference between fluoride sensitive and fluoride resistant SAPhase may relate to the varying association of a single enzyme with cell or lysosomal membranes. The close similarity of acid and neutral SAPhase suggests that both may represent a single enzyme in two forms rather than two distinct enzymes.     

Stability of the insoluble form of uridine kinase coupled to zn2+ or pb2+ ions.

Partially purified calf brain uridine kinase precipitated by bivalent metal cations has been compared with the soluble enzyme fraction regarding its stability in the presence of inactivating factors. The freeze-dried preparations of uridine kinase precipitaated by Pb2+ or Zn2+ ions, althouth enzymatically highly active, are insoluble in aqueous solutions. The activity of metal-insolubilized enzymes disappears during their preincubation in acidic media or in the presence of silver ions. Also trypsin, chymotrypsin and cathepsin B1 caused decreases in enzyme activity. However, fractions which have been precipitated by metal ions and freeze-dried are stable at high temperatures, whereas the activity of soluble uridine kinase is completely lost. Both unheated metal-ion precipitated uridine kinase preparations and those heated at 100 degrees C are equally sensitive to the feedback inhibition by CTP.     

Stable acid phosphatase: II. Effects of pH and inhibitors

Summary Microdensitometry demonstrated that stable acid phosphatase (SAPhase) in rat and hamster osteoclasts, chondroclasts, and chondrocytes has very similar properties. The differences that were observed suggest that conformational alterations in the enzymes may be responsible for inhibition by some agents such as tartrate. These differences in response to inhibitors depend on the method of embedding as well as on species differences. SAPhase appears to correspond to acid nitrophenyl phosphatase, as shown by its pH dependent re-activation, resistance to fluoride inhibition at nearneutral pH, and the inverse effect of pH on inhibition by zinc versus aluminium ions. That proportion of SAPhase resistant to fluoride is an acid phosphatase with activity at near-neutral pH rather than a strict neutral phosphatase. The difference between fluoride sensitive and fluoride resistant SAPhase may relate to the varying association of a single enzyme with cell or lysosomal membrances. The close similarity of acid and neutral SAPhase suggests that both may represent a single enzyme in two forms rather than two distinct enzymes.Supported by the Medical Research Council of Great Britain     

Adenylate cyclase cytochemistry: a methodological evaluation

Synopsis A cytochemical method for the demonstration of adenylate cyclase activity has been evaluated. Enzyme activity in the epithelium of the Müllerian part of the vaginal anlage in neonatal, oestradiol-treated mice has been studied under different experimental conditions. The effects of fixation, incubation conditions and post-fixation have been studied. Variations in the amount of impurities and acid content of the glutaraldehyde do not seem to influence the enzyme activity. High Pb²⁺ ion concentration seems to promote unspecific staining. Under standard conditions [2 mM Pb (NO₃)₂, pH 7.2, and incubation temperature 30°C], neither non-enzymatic nor nonsubstrate-dependent lead trapping in the tissue could be observed. The possible contribution of other enzymes utilizing ATP and AMP-P(NH)P as the formation precipitate, has been evaluated. Both ATP and AMP-P(NH)P have been used as substrates in this study. Provided the appropriate control experiments are performed, this cytochemical method is reliable for demonstration of adenylate cyclase activity.     

Cytochemical and autoradiographic studies of the localization and turnover of chromatin and nucleolar associated phospholipids in plant and animal tissues

The localization of chromatin-associated phospholipids has been demonstrated on chromosomes and on chromatin of interphase nuclei by cytochemical methods either in plant and in animal tissues. Three methods of fixation are suggested which can be combined which two cytochemical methods for phospholipids detection. An additional method is represented by autoradiographic technique after incorporation of a radioactive precursor such as [3H] ethanolamine. This method has been used with good results in nuclei of lateral root apices of Vicia faba on 1 micron thick section, thus avoiding any possible contamination by nuclear membrane. In these nuclei the phospholipids appear associated with the chromatin and more intensely with the nucleolar regions. The positivity of the cytochemical reaction disappears after extraction of fixed tissue with acidified methanol chloroform and after treatment of unfixed sections with phospholipase D. The use of phospholipid precursor has allowed the study of chromatin-phospholipids synthesis in root apices of Vicia faba with respect to timings of the cell cycle. The results show that there is a strong case for a pattern of chromatin phospholipid synthesis which operates during S phase. Concerning the role of phospholipids it is suggested that they may be linked to acidic protein and may have a structural function, particularly on the nucleoli.     

Isorenin, pseudorenin, cathepsin D and renin. A comparative enzymatic study of angiotensin-forming enzymes

1. Renin was purified 30 000-fold from rat kidneys by chromatography on DEAE-cellulose and SP-Sephadex, and by affinity chromatography on pepstatinyl-Sepharose. 2. The enzymatic properties of isorenin from rat brain, pseudorenin from hog spleen, cathepsin D from bovine spleen, and renin from rat kidneys were compared: Isorenin, pseudorenin and cathepsin D generate angiotensin from tetradecapeptide renin substrate with pH optima around 4.9, renin at 6.0. With sheep angiotensinogen as substrate, isorenin, pseudorenin and cathepsin D have similar pH profiles (pH optima at 3.9 and 5.5), in contrast to renin (pH optimum at 6.8). 3. The angiotensin-formation from tetradecapeptide by isorenin, pseudorenin and cathepsin D was inhibited by albumin, alpha-and beta-globulins. These 3 enzymes have acid protease activity at pH 3.2 with hemoglobin as the substrate. Renin is not inhibited by proteins and has no acid protease activity. 4. Renin generates angiotensin I from various angiotensinogens at least 100 000 times faster than isorenin, pseudorenin or cathepsin D, and 3000 000 times faster than isorenin when compared at pH 7.2 with rat angiotensinogen as substrate. 5. The 3 'non-renin' enzymes exhibit a high sensitivity to inhibition by pepstatin (Ki less than 5.10(-10) M), in contrast to renin (Ki approximately 6-10(-7) M), at pH 5.5. 6. It is concluded from the data that isorenin from rat brain and pseudorenin from hog spleen are closely related to, or identical with cathepsin D.     

In vitro effect of metal ions on the activity of two amphibian glyceraldehyde-3-phosphate dehydrogenases: potential metal binding sites

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was purified from two amphibian species, Xenopus laevis and Pleurodeles waltl. Comparative studies revealed that the two proteins differ by their subunit molecular masses, pI values and V8 digested peptide maps. The effect of zinc, cadmium and copper ions on GAPDH enzymatic activity has been examined in vitro. A time, metal concentration and metal type dependent inhibition was observed for both enzymes. X. laevis and P. waltl GAPDHs exhibit a much greater sensitivity to copper than to cadmium or zinc ions. Different half-lives and differential sensitivity to various metals was observed between the two enzymes with P. waltl GAPDH being remarkably tolerant to cadmium ions compared to the X. laevis enzyme. In order to understand the differential sensitivity of the two enzymes to metals, we produced 3D models of both X. laevis and P. waltl GAPDH structures based upon known 3D structures of GAPDHs from other species. This necessitated, in a first step, to clone a 900 bp cDNA fragment encoding the nearly full-length P. waltl GAPDH. Spatial motif searches on the homology models indicated potential metal binding sites involving cysteine and histidine residues outside the catalytic sites, existing only in either the X. laevis or the P. waltl GAPDH sequences.     

Cytochemical localization of adenylate cyclase and of calcium ion, magnesium ion-activated ATPases in the dense tubular system of human blood platelets.

Cytochemical techniques have been employed to study the localization of adenylate cyclase and (Ca2+ + Mg2+)-stimulated ATPase activities in platelets after fixation. Biochemical analysis of adenylate cyclase demonstrated a 70% reduction in activity in homogenates from fixed cells, but the residual activity could be stimulated 10--20 times by prostaglandin E1 (1 micrometer) under the same incubation conditions as employed in the cytochemical studies (e.g. media containing 2 mM lead nitrate and 10 mM NaF). Adenylate cyclase activity employing 5'-adenylyl-imiodiphosphate (AMP-P(NH)P) as substrate was found to be associated with the dense tubular system (smooth endoplasmic reticulum) in intact fixed platelets, and was apparent only when the cells were incubated with prostaglandin E1. Less activity was found along the membranes of the surface connected open canalicular system and occasionally at the outer cell surface. Enzymatic activity was blocked by the adenylate cyclase inhibitor 9-(tetrahydro-2-furyl) adenine and was not due to AMP-P(NH)P phosphohydrolase activity. The low adenylate cyclase activity in the surface membranes may be due to enzyme inactivation as a result of fixation, since a surface membrane fraction obtained by the glycerol lysis technique from unfixed cells had an adenylate cyclase specific activity equivalent to that in the microsomal membrane fraction. (Ca2+ + Mg2+)-stimulated ATPase activity was found associated with the membranes of the surface connected open canalicular system in unfixed cells. After brief fixation (5--15 min) with glutaradehyde, strong (Ca2+ + Mg2+)ATPase activity became apparent in the dense tubular system. Longer periods of fixation inactivated enzymatic activity. Addition of Ca2+ (1.0 mM) to incubation medium with low Mg2+ (0.2 mM), or increasing Mg2+ to 4.0 mM, in both cases strongly stimulated enzyme activity. The ATPase activity in the platelet membranes was not inhibited by ouabain. It is suggested that the Ca2+-stimulated ATPase and adenylate cyclase activities in the dense tubules may possibly be involved in regulation of intracellular Ca2+ transport.     

Some parameters affecting the activity of monoamine oxidase in purified bovine brain mitochondria.

Abstract— Some parameters affecting the activity of monoamine oxidase (MAO) in purified beef brain mitochondria were investigated, and diversities in enzyme properties were found as a function of substrate. The deamination of the biogenic amines: serotonin, dopamine, tyramine, tryptamine, phenylethylamine and two non-physiological amines, kynuramine and m-iodobenzylamine, was studied. Anions in high concentrations inhibited enzyme activity with kynuramine being the substrate most affected. Among the biogenic amines, the activity with the indolalkylamines showed greater sensitivity to mono-valent anions such as chloride than to polyvalent ions such as phosphate whereas the opposite was true with the phenylalkylamines. However, pyrophosphate ion had little or no effect on MAO activity, regardless of substrate. The inhibition of kynuramine and serotonin deamination was non-competitive but mixed competitive inhibition was found with tyramine and phenylethylamine. The activity of MAO was markedly affected by pH, and it had been previously reported that the substrates showed different pH optima in their oxidation. The effect of pH on activity has been attributed in part to changes in the ionization of the substrate and the hypothesis that the true substrate is the non-protonated amine. This was reflected in kinetic studies showing high substrate inhibition with increased pH. It was calculated that phenylethylamine would have the highest percentage of un-ionized amine at pH 8.2 and 9.1. At these pHs, there was more pronounced inhibition with high substrate concentrations of phenylethylamine than with the other substrates. In contrast, there was little inhibition with high substrate concentrations of tyramine which was the most ionizable of the substrates tested. When K_m values obtained at pH 7.4, 8.2 and 9.1 were corrected for ionization of the substrate, the corrected K_m was lowest at pH 7.4 for all substrates. Less than 50% of MAO activity was lost when beef brain mitochondria was heated at 50°C for 20 min. However, there was only a slight variation with substrate in the thermal inactivation experiments. It is concluded that the mitochondrial membrane environment surrounding the enzyme imposes certain restrictions on the enzymatic activity with respect to the different substrates which, in turn, are also affected by such parameters as pH and ions. The results are discussed in terms of the relationship of these factors to the question of enzyme multiplicity.     

Glycosyltransferases in plant and animal tissues effects of fixation and lead on enzyme activity.

As the initial step toward the cytochemical localization of glycosyl-transferases in situ, biochemical determinations of these enzyme activities from onion root tips and L1210 cells were performed before and after fixation as well as in the presence of lead ions. Glycosyltransferase activity from roots fixed in buffered formaldehyde or glutaraldehyde before homogenization decreased as the concentration of the fixative or fixation time was increased. Formaldehyde fixation was less inhibitory than glutaraldehyde; 35% of the glycosyltransferase activity was retained after 30 min fixation in 2% formaldehyde while 25% of the enzyme activity remained after a similar fixation in glutaraldehyde. Substantially higher levels of L1210 cell glycosyltransferase activity were retained after a 30 min 2% formaldehyde fixation (60% sialyltransferase; 82% galactosyltransferase), but inhibition by glutaraldehyde was similar to that observed for onion root galactosyltransferase. Glycosyltransferase from formaldehyde-fixed roots was inhbited 35% by lead nitrate, but sialytransferase from formaldehyde-fixed L1210 cells was unaffected by lead ions. These findings are encouraging for further studies aimed at the development of cytochemical technique to localize glycosyltransferase in plant and animal tissues.     

Finding stable cellulase and xylanase: evaluation of the synergistic effect of pH and temperature

Ethanol from lignocellulosic biomass has been recognized as one of the most promising alternatives for the production of renewable and sustainable energy. However, one of the major bottlenecks holding back its commercialization is the high costs of the enzymes needed for biomass conversion. In this work, we studied the enzymes produced from a selected strain of Aspergillus niger under solid state fermentation. The cellulase and xylanase enzymatic cocktail was characterized in terms of pH and temperature by using response surface methodology. Thermostability and kinetic parameters were also determined. The statistical analysis of pH and temperature effects on enzymatic activity showed a synergistic interaction of these two variables, thus enabling to find a pH and temperature range in which the enzymes have a higher activity. The results obtained allowed the construction of mathematical models used to predict endoglucanase, β-glucosidase and xylanase activities under different pH and temperature conditions. Optimum temperature values for all three enzymes were found to be in the range between 35°C and 60°C, and the optimum pH range was found between 4 and 5.5. The methodology employed here was very effective in estimating enzyme behavior under different process conditions.     

Effect of triiodothyronine on the activity and sensitivity of glycosidases to heavy metals (Cu,Zn, and Pb) in juvenile blue bream <Emphasis Type="Italic">Ballerus ballerus</Emphasis> (L.)

The influence of exogenous triiodothyronine (0.25 ng/mL) on the activity glycosidases (maltase, amylolytic activity) and their sensitivity to Cu, Zn and Pb ions (25 mg/L) has been studied in the whole organism of juvenile blue bream Ballerus ballerus (L.). We have found that the treatment of fish with triiodothyronine resulted in an increase in amylolytic activity. Maltase activity is not affected. In addition, the exposure to exogenous triiodothyronine results in a decrease in the sensitivity of glycosidase to the in vitro action of the ions of biogenic metals Cu and Zn and in an increase in the sensitivity of the maltase to the action of Cu, Zn, and Pb ions.     

Cytochemical profile of immunoregulatory T-lymphocyte subsets defined by monoclonal antibodies

Immunogold staining in combination with enzyme cytochemistry was used to determine the cytochemical profile of the immunoregulatory T-lymphocyte subpopulations defined by the monoclonal antibodies OKT3, OKT4, OKT8, and OKM1 in normal peripheral blood. Leukocyte suspensions were first incubated with the monoclonal mouse antibodies and then with colloidal gold-labeled goat antimouse antibodies. The cells were fixed and cytocentrifuge preparations were made. Cytochemical reactions for the detection of peroxidase, acid alpha-naphthyl acetate esterase, acid phosphatase, and beta-glucuronidase were performed on these preparations. Under light microscopy, lymphocytes reacting with the monoclonal antibodies had numerous dark granules around their surface membrane. In the cytoplasm the intracellular enzymatic activities were stained. The T-lymphocytes were characterized by a dot-like activity for the three enzymes. No significant difference could be found between the cytochemical profile of the T-helper (OKT4 positive) and T-cytotoxic suppressor cell populations (OKT8 positive). A few cells with lymphocyte morphology reacted with the OKM1 monoclonal antibody. Their cytochemical characteristics were different from those of mature T-cells (OKT3 population) or mature monocytes. From the comparison of their cytochemical characteristics, we can conclude that there is little correlation between the immunoregulatory T-lymphocyte subsets defined by these monoclonal antibodies and those defined by Fc receptors.