17-Hydroxyprogesterone metabolism in the monkey fetal adrenal: C17–20Lyase and 21-hydroxylase activities

C^17–20Lyase and 21-hydroxylase activities were measured during late gestation In the rhesus monkey ($\text{Macaca mulatta}$) fetal adrenal. Activities were assessed in 10,000 × g supernatants with 17-hydroxyprogesterone and NADPH as substrates. Although conversion of [¹⁴C]17-hydroxyprogesterone to [¹⁴C]androstenedione was noted, activity was often nonlinear and far less than the rate of hydroxylation which together prevented an accurate estimation of lyase rate, K_m and V_max. 21-Hydroxylase activity was characterized; the mean reaction rate was 1.6 × 10^?3 μmoles NADPH oxidized/min. × mg^?1 protein with an apparent K_m of 3.6 × 10^?7 M and a V_max of 2.2 × 10^?3 μmoles/min. × mg^?1 protein. These values were similar to data obtained In adrenals from adult monkeys. A relatively high level of hydroxylase activity in the fetal gland might lead to an Inadequate supply of precursors for the synthesis of dehydroepiandrosterone sulfate (DHEAS) in the adrenal if it also contained 3β-hydroxysteroid dehydrogenase (3β-hsdh). However, the fact that the fetal adrenal reportedly is deficient in 3β-hsdh may serve to protect both DHEAS and corticoid synthesis.     

Arachidonic acid, bradykinin and phospholipase A2 modify both prolactin binding capacity and fluidity of mouse hepatic membranes

The objective of this study was to determine if arachidonic acid, a precursor of prostaglandin synthesis, bradykinin, a decapeptide known to stimulate membrane phospholipid methylation, arachidonic acid release and prostacyclin synthesis, and enzyme phospholipase A₂, capable of liberating arachidonic acid, alter the fluidity of hepatic membranes which could in turn modify the functionality of prolactin receptors. Liver homogenates of adult C₃H female mice incubated at 28°C for various times with 1–20 μg/ml arachidonic acid, 1–100 μg/ml bradykinin or 0.26–0.00026 U/ml phospholipase A₂ provided the 100,000 × g membrane pellets for subsequent ovine prolactin binding and membrane fluidity studies. Membrane microviscosity was determined by fluorescence polarization techniques using the lipid probe 1,6 diphenylhexatriene. Arachidonic acid, bradykinin and phospholipase A₂ stimulated specific oPRL binding, in a dose-related fashion, with maximum increases of 73%, 21% and 46%, at 4 μg/ml arachidonic acid, 5 μg/ml bradykinin and 0.026 U/ml PLA₂, respectively. This induction, occurring within 30 min of incubation, was found to be due to an increase in the number of receptor sites. Under the same conditions, arachidonic acid, bradykinin and PLA₂ induced 22%, 16%, and 18% decreases in membrane microviscosity, respectively. These data suggest that prostaglandin synthesis modifying agents may modulate the number of prolactin receptors $\text{in vivo}$ by changing the lipid fluidity of the target cell membranes by either of their known effects: arachidonic acid release from the phospholipid matrix, synthesizing appropriate prostaglandins at correct concentration or methylation of membrane phospholipids.     

Bioreduction of nitroxides by Staphylococcus aureus.

Two nitroxide radicals (TEMPO, I; OXAN, II) and a spin labeled penicillin (III) were reduced by $\text{Staphylococcus aureus}$. A short induction period preceded zero order reduction of these substrates leading to a K_m of 8 × 10^?4M, 6.67 × 10^?5M and 5.7 × 10^?4M and V_max of 106, 26 and 11 μ mole/min mg bacteria for I, II and III, respectively.     

Kinetic parameters for the binding of p-nitrophenyl alpha-d-mannopyranoside to concanavalin A.

Binding of the chromogenic ligand p-nitrophenyl α-d-mannopyranoside to concanavalin A was studied in a stopped-flow spectrometer. Formation of the protein-ligand complex could be represented as a simple one-step process. No kinetic evidence could be obtained for a ligand-induced change in the conformation of concanavalin A, although the existence of such a conformational change was not excluded. The entire change in absorbance produced on ligand binding occurred in the monophasic process monitored in the stopped-flow spectrometer. The value of the apparent second-order rate constant (k_a) for complex formation (k_a = 54,000 s^?1m^? at 25 °C, pH 5.0, Γ/2 0.5) was independent of the protein concentration when the protein was in the range of 233–831 μm in combining sites and in excess of the ligand. The apparent first-order rate constant (k_?a) for dissociation of the complex was obtained from the rate constant for the decomposition of the complex upon the addition of excess methyl α-d-mannopyranoside (k_?a = 6.2 s^?1 at 25 °C, pH 5.0, Γ/2 0.5). The ratio $\text{k}{}_{\mathrm{<mtext>a</mtext>}}{}_{\mathrm{<mtext>?</mtext><mtext>a</mtext>}}$ (0.9 × 10₄m^?1) was in reasonable agreement with value of 1.1 ± 0.1 × 10⁴m^?1 determined for the equilibrium constant for complex formation by ultraviolet difference spectrometry. Plots of ln($\text{k}{}_{\mathrm{<mtext>a</mtext>}}\text{T}$) and ln($\text{k}{}_{\mathrm{<mtext>a</mtext>}}\text{T}$) vs $\text{1}\text{T}$ were linear (T is temperature) and were used to evaluate activation parameters. The enthalpies of activation for formation and dissociation of the complex are 9.5 ± 0.3 and 16.8 ± 0.2 kcal/mol, respectively. The unitary entropies of activation for formation and dissociation of the complex are 2.8 ± 1.1 and 1.3 ± 0.7 entropy units, respectively. These entropy changes are much less than those usually associated with substantial changes in the conformation of proteins.     

Progressive increase with time after sensitization in the functional affinity of T lymphocytes

The dose response relationship in peritoneal cell migration inhibition, elicited by various concentrations of ABA-Tyr, was studied in guinea pigs sensitized with a standard dose of ABA-Tyr. The reactivity to 2 × 10^?7 or 2 × 10^?8 mole/ml appeared at 2 wk, and remained at the maximum level from 4 wk to 8 mo after sensitization. The inhibition by 2 × 10^?9 mole/ml increased up to 4 mo and 2 × 10^?10 mole/ml was first inhibitory at $\text{6}\text{1}\text{2}$ mo. The change in the slope of the dose response curve was a property of the ABA-specific cells. As there is no B-cell response to ABA-Tyr, the finding shows that the functional affinity of specific T cells progressively increases with time after sensitization.     

Mechanism of action of thrombin on fibrinogen. The reaction of thrombin with fibrinogen-like peptides containing 11, 14, and 16 residues

The following peptides were synthesized by classical methods in solution: Ac-Gly-Gly- Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-NHCH₃ (A), Ac-Ala-Glu-Gly-Gly-Gly-Val- Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-NHCH₃ (B), and Ac-Phe-Leu-Ala-Glu-Gly-Gly- Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-NHCH₃ (C). The rates of hydrolysis of the Arg-Gly bond of these three peptides by thrombin were measured, and the values of $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ were found to be 0.05 × 10^?7 (A), 0.02 × 10^?7 (B), and 1.6 × 10^?7 (C) [(NIH units/ liter)s]^?1. The value of$\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ for peptide C is less than 1% of that for fibrinogen [although the value of k_cat itself, for peptide C (but not for A or B), is comparable to that for fibrinogen]. These results indicate that phenylanine and leucine at positions P₉ and P₈, respectively, play a key role in the reaction of thrombin with fibrinogen. The data also show that factors outside of the 16 residues of peptide C are important in determining the rate of hydrolysis of fibrogen by thrombin.     

Regulation of high- and low-affinity epidermal growth factor receptors by glucocorticoids

It is reported that receptors for epidermal growth factor (EGF) in HeLa S₃ cells exist in two forms, which differ in both affinity and capacity. Both the number of receptors and their distribution into low- and high-affinity forms are modulated by glucocorticoids. Scatchard analysis of saturation binding assays performed at 0 °C indicates that there is a low-affinity class of receptors ($\text{K}{}_{\mathrm{d}}\text{? 1.5}\text{nm}$), which contains approximately 6 × 10⁴ binding sites per cell, and a second, high-affinity class of receptors ($\text{K}{}_{\mathrm{d}}\text{? 0.16}\text{nm}$) containing approximately 5 × 10³ binding sites per cell. Exposure of HeLa S₃ cells to 10^?7m dexamethasone for 24 h increased EGF binding to whole cells by increasing the numbers of low- and high-affinity receptors by 20 and 114%, respectively. The increase in EGF binding depends upon the dose of dexamethasone, being raised from 10^?11 to 10^?6m. EGF binding is half-maximal near 2–4 × 10^?9m, a concentration equal to the K_d of dexamethasone for the glucocorticoid receptor in these cells. The increase in EGF binding is specific for glucocorticoids, occurring when the HeLa S₃ cells are exposed to 10^?7m cortisol or dexamethasone for 24 h, but not when the cells are similarly treated with testosterone, 5α-dihydroxytestosterone, 17β-estradiol, or progesterone. The effect on EGF binding appears to be biphasic; the initial rapid increase occurs between 8 and 12 h, is blocked by both 10^?6m cyclohexamide and 0.1 μg/ml actinomycin D, and is followed by a more gradual increase thereafter. These data indicate that glucocorticoids are able to regulate both the number of EGF receptors and their distribution into high- and low-affinity components. Press, Inc.     

Properties of α-bungarotoxin binding sites in foetal human brain

Maximum levels of binding of α-bungarotoxin to foetal human brain membranes were found to remain essentially constant at 30–50 fmol/mg protein (1.1–1.5 pmol/g wet weight in whole brain) between gestational ages of 10 and 24 weeks. Equilibrium binding of α-bungarotoxin to both membranes and to detergent extracts showed saturable specific binding to a single class of sites with K_d (app) values of 3.5 × 10^?9 M and 2.4 × 10^?9 M respectively. Association rate constants, determined from time courses of binding of α-bungarotoxin to membranes and detergent extracts, were 2.3 × 10⁵ M^?1 sec^?1 and 2.6 × 10⁵ M^?1 sec^?1 respectively. Dissociation of α-bungarotoxin from both membrane and detergent extracts showed a rapid initial rate with $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ approx 15 min which, in the case of the detergent extract, was followed by a slower dissociation accounting for the remaining 20% of the bound ligand. Competition studies with a number of cholinergic ligands indicated that the α-bungarotoxin-binding sites in foetal brain display a predominantly nicotinic profile.     

Oxytocin receptors in rat oviduct.

Particles from rat oviduct homogenates sedimenting between 1,000 × g for 10 min and 48,000 × g for 30 min bound [³H]oxytocin $\text{in vitro}$. The apparent K_d for oxytocin binding to high affinity sites in particles prepared from estrogen-treated rats was 1.8 × 10^?9 M. About 215 fmoles of oxytocin were bound per mg of particulate protein. Oviducal preparations from untreated rats had about 25% the affinity for oxytocin of preparations from estrogen-treated rats. Oxytocin analogues were bound to oviducal particles in the same rank order as their uterotonic potencies: (desamino)oxytocin > (4-threonine)oxytocin > oxytocin > (8-lysine)vasopressin ? desaminotocinol. No oxytocin binding could be shown with the particulate fractions from rat ovary. The binding of oxytocin to the oviduct and uterus are similar in affinity, number of binding sites, ligand specificity, and the increase in response to estrogen treatment.     

Synthesis and biological activity of four gamma-melanotropin peptides derived from the cryuptic region of the adrenocorticotropin/beta-lipotropin precursor.

A third melanotropin coding fragment named γ-MSH was discovered by Nakanishi et al (Nature $\text{278}$, 423–427 (1979)) in the cryptic region outside the portion coding for ACTH and β-LPH in the ACTH/β-LPH precursor mRNA isolated from the intermediate lobe of bovine pituitary. Four possible γ-MSH peptides derived from this coding fragment were synthesized by solid-phase methodology and their bioactivity determined in an $\text{in vitro}$ MSH assay as well as the anterior pituitary primary culture assay. Relative to α-MSH, the melanotropic activities of Ac-γ₁-MSH, γ₁-MSH, γ₂-MSH and γ₃-MSH are 7.3 × 10^?4, 3.3 × 10^?5, 1.4 × 10^?4 and 4.6 × 10^?7 respectively. None of these γ-MSH peptides releases LH, FSH, PRL, GH and TSH in the pituitary culture medium at a dose as high as 100 ng per dish.     

Morphine release and displacement by naloxone in vivo in morphine naive and withdrawn rats

Male Long-Evans rats, implanted in the lateral cerebroventricle with chronic indwelling push-pull cannulae, were perfused (10 μl/min) for 120 min: 20 min with 1.5 × 10^?6M morphine in sterile isotonic saline containing 2.3 mM CaCl₂ (vehicle); 40 min with vehicle; 20 min with 1.5 × 10^?6M morphine; 10 min with vehicle and 30 min with 1 × 10^?6M naloxone in vehicle. These rats and drug-naive rats were implanted s.c. with 2 × 50 mg morphine pellets. After 72 hr the pellets were removed and 18–24 hr later the above perfusion procedure was repeated. The amount of morphine collected in the perfusate during the washout with naloxone was elevated, compared to the amount collected during the corresponding time of the washout with vehicle for both naive and withdrawn groups. The enhanced morphine release during the washout with naloxone did not differ significantly between the naive and withdrawn rats. However, significantly less morphine was recovered in the perfusate collected during the vehicle washout from the withdrawn rats, compared to that collected from the naive rats. The data suggest that $\text{in vivo}$ morphine is specifically bound to receptors and is sensitive to naloxone displacement. It is also concluded that morphine is differentially taken up or otherwise disposed of by brains of rats which are in opiate withdrawal.     

Relaxation kinetics of the helix-coil transition of a self-complementary ribo-oligonucleotide: A7U7

Relaxation measurements on the kinetics of the double helix to coil transition for the self-complementary ribo-oligonucleotide A₇U₇ are reported over a concentration range of 6.9 μM to 19.6 μM in single strand in 1 M NaCl. The rate constants for helix formation are about 2 × 10⁶ M^?1 s^?1 and decrease with increasing temperature yielding an activation enthalpy of ?6 $\text{kcal}\text{mole}$. The rate constants for helix dissociation range from 3 to 250 s^?1 and increase with increasing temperature yielding an activation enthalpy of +45 $\text{kcal}\text{mole}$. The kinetic data reported here for 1 M NaCl is compared with previously published results obtained at lower salt concentrations. These data are discussed in terms of the quantitative effect of ionic strength on the kinetics of helix-coil transitions in oligo- and polynucleotides.     

Tryptophan uptake by Mycobacterium tuberculosis H37Rv--inhibition by isoniazid.

The effect of various sub-inhibitory concentrations of isoniazid on tryptophan uptake by $\text{Mycobacterium tuberculosis}$ H₃₇Rv grown $\text{in vitro}$ and $\text{in vivo}$ was studied. Uptake, measured after 3 minutes of drug exposure was inhibited mildly by 0.1 μg/ml and 0.2 μg/ml concentration and completely by 0.3 μg/ml. However, with the minimal inhibitory concentration (MIC)⁷ of 0.5 μg/ml, not only inhibition but also a strong efflux of the preformed tryptophan pool were observed. The results are discussed in the light of the theory that isoniazid interferes with the cell wall mycolate synthesis.     

Photodynamic alteration of cornea endothelium: Relation to bicarbonate fluxes and oxygen concentration

Corneas were mounted in flux chambers and endothelial bicarbonate fluxes were determined following sensitization of endothelial cells with 5 · 10^?6 M rose bengal and exposure to light. Corneas exposed to light demonstrated an increased passive bicarbonate flux compared to corneas not photosensitized. Active bicarbonate flux was reduced after 5 min of light exposure, but not after 1 min of light exposure. The increase in passive bicarbonate flux was prevented by the addition of 200 μg/ml catalase to the bathing solution; however, catalase had no effect on the photodynamic alteration of active flux. Neither 10 mM ascorbic acid nor 1.012 g/l glutathione prevented the photodynamically induced increase in passive flux. Perfusion of corneas with 5 · 10^?6 M rose bengal dissolved in a sucrose-substituted Krebs-Ringer bicarbonate solution with a $\text{p}{}_{\mathrm{<mtext>o</mtext>}}{}_2$ of $\text{124 ± 4.0}\text{mmHg}$ and exposed to light swelled at rates more rapid than corneas treated in a similar fashion but perfused with a solution with a $\text{P}{}_{\mathrm{<mtext>o</mtext>}}{}_2$ of $\text{20 ± 4.6}\text{mmHg}$. This study demonstrates that photodynamically induced corneal endothelial cell alteration results in increased passive bicarbonate flux, a time-dependent decrease in active bicarbonate flux, is oxygen dependent, and is at least in part secondary to H₂O₂ produced by the dismutation reaction of the superoxide free radical.     

Potentiation of bradykinin-induced vascular permeability increase by prostaglandin E2 and arachidonic acid in rabbit skin

The activity of prostaglandins (PG) in producing vascular permeability was quantitated by dye extraction method in skin of anaesthetized rabbits. PGE₁ and PGE₂ (0.01–10 μg) produced increase in vascular permeability. Activity was approximately equal to that of histamine (Hist) and $\text{1}\text{20}$ of that of bradykinin (BK) on a weight basis. The activity of PGF_1α and PGF_2α was only $\text{1}\text{20}$ of that of PGE₁ or PGE₂.In spite of the relatively low potency of PGE₁ and PGE₂ in the rabbit, near threshold doses (0.1 or 1 μg) of PGE₂ could potentiate permeability responses to bradykinin (0.1 μg) by 10 or 100-fold, respectively. Equivalent doses (0.1 or 1 μg) of histamine could not potentiate the bradykinin responses. Arachidonic acid (AA) at 1 μg, produced a 10-fold potentiation in the permeability response to bradykinin (0.1 μg). Pretreatment of the rabbits with indomethacin (20 mg/kg, i.p.) reduced the responses of BK (0.1 μg) + AA (1 μg) down to a similar magnitude of those seen with bradykinin alone. However, indomethacin did not block responses to either, BK alone, BK + PGE₂, or BK + Hist. Various doses (1, 10, 100 and 300 μg) of arachidonic acid alone also produced increase in cutaneous vascular permeability, although its potency was only $\text{1}\text{3}$–$\text{1}\text{8}$ of that of PGE₂. This activity of arachidonic acid was attributed in part to its bioconversion to PGE₂, since its activity was significantly reduced by the prostaglandin antagonist, diphloretin phosphate (DPP) (60 mg/kg, i.v.) and by indomethacin (20 mg/kg, i.p.), which blocks conversion of arachidonic acid to prostaglandins. Arachidonic acid may owe some of its permeability increaseing effects to histamine release, since its effects were also reduced by the antihistamine, pyrilamine (2.5 mg/kg, i.v.).     

Actions of morphine on prostate gland fructose metabolism

Varying doses of morphine sulfate (10, 20 or 40 mg/kg daily × 10) were observed to suppress metabolic activities in the mouse prostate gland. Prostate gland fructose, an index of androgenic activity, was significantly reduced by these dose regimes of morphine (P < 0.01). Injections of morphine sulfate (20 mg/kg daily × 10) led to an inhibitition in the $\text{in vitro}$ synthesis of both fructose^?14C and sorbitol^?14C from glucose^?14C by the prostate gland, part of which may have been due to decreased uptake of glucose by the gland. The $\text{in vitro}$ assimilation of 2-deoxyglucose^?14C by the prostate was also reduced by morphine treatment. The $\text{in vitro}$ actions of morphine (2 × 10^?3M) on the metabolism of radioactive glucose by the mouse prostate gland likewise revealed a significant reduction in the formation of sorbitol^?14C, but no decrease in fructose^?14C formation. These results indicate that both the $\text{in vitro}$ and $\text{in vivo}$ actions of morphine can inhibit fructose metabolism in the prostate gland.     

N-acetyl-(L-Ala) 3 -p-nitroanilide as a new chromogenic substrate for elastase

The introduction of a useful new chromogenic substrate for the determination of elastase (EC 3.4.4.7) activity is described. N-acetyl-L-Ala-L-Ala-L-Ala-$\text{p}$-nitroanilide (AcAla₃NA) is a new specific elastase substrate whose hydrolysis can be followed spectrophotometrically at 410 nm in a wide pH range. Its rate of hydrolysis by α-chymotrypsin (EC 3.4.4.5) and trypsin (EC 3.4.4.4.) is 0.02% and 0.001% respectively compared to its rate of hydrolysis by elastase. As little as 0.1 μg elastase/ml can be satisfactorily determined. At pH 8, K_m = 0.88 mM and k_cat = 11.9 sec^?1.     

Stereospecific interaction of opiate narcotics in binding of 3H-dihydromorphine to membranes of rat brain

Membranes from homogenates of corpus striatum bound ³H-dihydromorphine in a saturable fashion with a Km value of 1 × 10^?9M. The binding of ³H-dihydromorphine to the membranes was reduced to about 10% by 10^?7M levorphanol but not by 10^?7M dextrorphan. The binding of ³H-dihydromorphine became less sensitive to 10^?7M levorphanol when the concentration of ³H-dihydromorphine was greater than 2 × 10^?9M. Other opiate narcotics, e.g. morphine and $\text{l}$-methadone, were as effective as levorphanol in competition for the binding ³H-dihydromorphine with ED₅₀ values of 2–4 × 10^?9M. $\text{d}$-Methadone and dextrorphan were about 1/50 and 1/2000 as effective as their respective levo-isomers. The opiate antagonist, naloxone, also competed effectively for the binding sites with an ED₅₀ value of 3.3 × 10^?9M. Substances like acetylcholine, choline, serotonin, norepinephrine and dopamine were ineffective. Only ionophores specific for divalent cations stimulated the binding of ³H-dihydromorphine suggesting that some endogenous divalent cations may be inhibitory to the binding of the opiate narcotic. The receptors of ³H-dihydromorphine probably exist in the membranes of nerve endings and have a density of 6 × 10¹² sites per g in corpus striatum. We conclude that the described technique can successfully detect the opiate narcotic receptors in the central nervous system without the usual method of displacement.     

Mechaism of Arthrobacter sialophilus neuraminidase: The binding of substrates and transition-state analogs

2-Deoxy-2,3-dehydro-N-acetylneuraminic acid and its methyl ester are competitive inhibitors of Arthrobacter sialophilus neuraminidase with K_i = 1.4 × 10^?6$\text{M}$ and 4.8 × 10^?5$\text{M}$, respectively. The K_m for the substrate, N-acetylneuraminlactose, is 1.0 × 10^?3$\text{M}$. These data, taken together with the conformation of these compounds, indicate that these compounds are transition-state analogs of the enzyme. These results also suggest that the substrate upon binding to neuraminidase is distorted to a conformation approaching that of a half-chair.     

Catalytic effect of sulfhydryl oxidase on the formation of three-dimensional structure in chymotrypsinogen A

The effect of sulfhydryl oxidase on the rate of disulfide bond formation and polypeptide chain folding in reductively denatured chymotrypsinogen A has been investigated using an immobilized zymogen preparation and a cylindrical quartz flow-through fluorescence cell. Enzymatic oxidation of the 10 sulfhydryl groups in reduced chymotrypsinogen followed first order kinetics at pH 7.0 with an apparent first order rate constant governing sulfhydryl group disappearance of 4.2 × 10^?2 min^?1. This provides a $\text{t}\text{1}\text{2}$ of 16.3 min for the sulfhydryl oxidase-catalyzed oxidation, whereas 165 min are required for nonenzymatic aerobic oxidation of one-half the sulfhydryl groups. Refolding of the reductively denatured polypeptide chains, monitored by changes in protein fluorescence, did not follow first order kinetics characteristic of a simple two-state mechanism, nor did the return of trypsin activatability. It appears that at least one intermediate must exist in such refolding, in both the uncatalyzed and sulfhydryl oxidase-catalyzed processes. Estimation of the rate constants governing refolding, assuming a single intermediate between the denatured and native states, provided values of 3 × 10^?2 min^?1 and 7 × 10^?3 min^?1 for uncatalyzed autoxidation and 4 × 10^?2 min^?1 and 1.1 × 10^?2 min^?1 for the sulfhydryl oxidase-catalyzed transition. Thus, enzymic catalysis of disulfide bond formation can lead to apparent catalysis of protein refolding as monitored both by fluorescence and by acquisition of biological function.