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Finally, the cells were suspended in the refrigerated HBE buffer at 1: Pooled buffy coats having a mean count of 8. This yield corresponded to an enrichment of approximately 2. Cells were treated with apyrogenic solutions and using sterile disposable plastic ware, and kept on ice to prevent any spontaneous activation [ 30 ]. We only included experiments that met the following criteria: Applying these criteria, five of the seven experiments performed with histamine dilutions and five of the six experiments performed with water dilutions were selected as valid.

The negative controls consisted of isotype matched, directly conjugated non-specific antibodies. Supernatants were removed and the pellets gently suspended in 0. The threshold between CD63 non-expressing and CD63 expressing basophils was arbitrarily set in each experiment close to the right-hand limit of the fluorescence peak of a sample of unstimulated, untreated cells.

The MFI was calculated by the cytometer software by averaging the total fluorescence of the marker in the basophil gate. The observed MFI values for each experiment were recorded, and exploratory statistics such as the average and standard deviation were computed. Normality was checked by the Shapiro—Wilk test. The symmetry of the data was also tested, showing our experimental MFI values to be strongly right-skewed.

In light of the non-symmetrical distribution of the data, which clearly did not satisfy the assumption of normality, it was decided to use non-parametric tests. Since ANOVA was unsuitable due to non-normality, for the global analysis of all the treatments, we adopted the Friedman non-parametric test for multiple-related samples. For the comparison between control and treatment groups, we applied the two-tailed Wilcoxon non-parametric test for paired samples.

The data were first of all paired by experiment: We then took all the 15 values from the five experiments on each treatment group and compared them with all the 15 values from the control no histamine group. Since the CDc is constitutively expressed at low level in resting, unstimulated basophils, the net activation response to anti-IgE dMFI was calculated by subtracting the mean resting fluorescence expressed by unstimulated cells in each experiment from the fluorescence values of anti-IgE activated samples.

Then, the percentage inhibitory effect of each dilution on the cell activation to the anti-IgE was calculated, for each experiment, according to the formula:. Basophil response toward the agonist was initially examined by evaluating two-parameter dot plots of the acquired events Fig. Non-activated resting basophils Fig. From these two-parameter dot plots, a large population of activated basophils expressing a CDc bright phenotype was clearly evident, while most of these activated cells were negative at the CD63 staining.

This confirms that CDc is a more suitable type of marker for this type of study [ 17 , 28 ], particularly when the extent of activation is low [ 26 ]. Activation of basophils with anti-IgE caused a considerable increase in the MFI of CDc compared with unstimulated cells from a mean of The complete series of 15 replicates was analyzed using non-parametric tests since the distribution of values was not normal.

Histamine 2C positive control reduced the response to CDc expression in human basophils after the specified treatments, measured as mean fluorescence intensity units MFI. The response of control, uninhibited samples to anti-IgE shifted from a mean of However, none of the water succussed dilutions showed any significant effect on the test system in two-sample comparisons with uninhibited, anti-IgE-activated sample.

The percentage of spontaneous activation no anti-IgE was very low 1. Following challenge with anti-IgE, the CD63 expressing cells increased to 8. This difference between these two series of experiments, in the basophil response to anti-IgE could be explained by the very low expression of CD63 in our experimental conditions see also Fig. A further factor of variability could be related to high inter-individual differences of basophil responsiveness, possibly due to seasonal variations or previous contacts with sensitizing substances, previously noted by others [ 27 , 33 ], which may be reduced but not completely eliminated by pooling buffy-coats from different donors.

Anymore, also with this marker all the cells tested were strongly inhibited by histamine at low dilution 2C , which decreased the CD63 expressing cells to 4. A few high histamine dilutions also decreased the percentage of CD63 expressing cells e.

No effects were determined by highly diluted water and succussed solutions. CD63 expression in human basophils after the specified treatments, measured as percentage of cells which up-regulated CD Preliminary experiments showed that histamine low and high dilutions did not affect the resting MFI but only the activation response.

So the percentage effect was calculated not on the absolute values, but only on the net MFI after subtracting the resting MFI. Histamine 2C positive control inhibition was There was no significant difference between the inhibition by histamine 2C in the two series. The dilution—effect histogram of histamine A shows a non-linear behaviour, with peaks at 14C and 16C dilution and paradoxically less activity at lower dilutions. No significant effects of water control dilutions B were observed.

Percentage inhibition of anti-IgE-activated CDc expression in human basophils treated with high histamine dilutions a or with high water dilutions b. CDc MFI values of the uninhibited activation were Human basophils are peripheral granulocytes involved in immune and inflammatory processes such as hypersensitivity and allergic reactions.

Among the various soluble factors able to regulate basophil function, a considerable body of literature has been devoted to histamine, a well-known mediator of inflammation produced by basophils and mast cells. Histamine at high doses acts as a downstream regulator by interacting with H2-receptors, thus inhibiting basophil release of mediators and degranulation [ 36 ]; it is therefore very intriguing that the same effect was observed with highly diluted histamine but not with highly diluted histidine [ 11 ], and that it could be inhibited by the H2-antagonist cimetidine [ 37 ].

Multicentre analyses have partially confirmed these findings [ 10 ], but other groups also report some negative results [ 19 ]. In this work we were able to reproduce evidence for the effects of high histamine dilutions on anti-IgE-triggered basophil activation, specifically evaluated by the CD MFI, using a standardized approach with the following characteristics: The resultant dose—effect curve displayed a very unusual behaviour, whose underlying mechanism remains to be investigated.

It is worth noting that the range of active dilutions is very close to the 16C dilution reported elsewhere [ 11 , 15 ], although Brown and Ennis [ 13 ] instead reported inhibitory activities for 10C and 13C, but not for the 12C, 14C and 16C dilutions. However, this discrepancy may be interpreted as due to the lower sensitivity of this parameter to inhibition by high dilutions or otherwise as a result of the high variability of this response, because of the low percentage of cells expressing CD63 in our test system.

As a consequence, the high-dilution effects were much more clearly and consistently observable by evaluating the CDc marker. We have previously shown that the chemotactic peptide fMLP, at very low dose, causes up-regulation of CDc but not of CD63 [ 26 ], and this observation is consistent with the finding that CDc reflects the priming events triggered by interleukin-3, which occur previously to degranulation events [ 28 ].

Moreover, CDc expression in not inhibited by phosphatidylinositolkinase inhibitors, as is CD63 [ 29 ]. Thus, small changes in the experimental conditions used or subtle distinction of the signal transduction mechanisms of the two markers expression could differentiate the results of high dilution experiments obtained by different authors in this model.

Research on extremely sensitive systems and very high dilutions of substances suggests that trace elements, as well as container materials, storage durations and shaking methods, may influence the results [ 39 ]. Therefore, suitable water controls prepared in an identical manner and subjected to the same storage time should be used.

These considerations, coupled with the highly controversial nature of the phenomenon, which would have significant pharmacological implications but is often judged to be improbable and implausible from a conventional scientific perspective [ 24 , 40 , 41 ], make it all the more important that more replications should be done independently to establish models that are stable across laboratories and teams [ 22 ]. The possible biological mechanism s underlying the regulatory processes affected by high histamine dilutions remain to be elucidated.

These findings suggest that the high dilutions of histamine, which are better observed through CDc expression, might affect some subtle and early level of signal transduction, similar to the priming effects of very low doses, instead of causing a general inhibition of cell responses. Further studies are needed to confirm whether this hypothesis is applicable to the high-dilution effects observed on human basophils.

So far there is no satisfactory or uniting theoretical explanation for these observations, but recent evidence seems to point to organization of the solvent water on a mesoscopic scale: It was suggested that histamine molecules might act as nucleation centres, amplifying the formation of stable supramolecular structures, involving nanobubbles of atmospheric gases and highly ordered water around them.

In the future, the possible existence and the nature of clathrate-like hydrate nanostructures formed during the dilution and succussion process might be explained by cluster science, in which different geometrical structures of clusters composed of the same chemical species may differ in their chemical reactivity [ 50 ]. These unusual properties of high dilutions, which merit further investigation, are potentially relevant not just to homeopathic pharmaceutical practice, but also to basic research into cell sensitivity to regulation.

The authors wish to thank Dr Luigi Marrari for his advice and cooperation. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author s and source are credited. National Center for Biotechnology Information , U. Published online May 6. Author information Article notes Copyright and License information Disclaimer.

This article has been cited by other articles in PMC. Abstract Objective Previous research suggests that human basophil activation may be inhibited by histamine even at extremely low doses high dilutions.

Cell preparation Basophil-enriched cell samples were prepared by pooling the leukocyte buffy coats drawn from 3-ml venous samples of K 2 -ethylendiaminotetraacetic acid EDTA anti-coagulated peripheral blood of four healthy, non-allergic, subjects blood donors. Statistics The observed MFI values for each experiment were recorded, and exploratory statistics such as the average and standard deviation were computed. Then, the percentage inhibitory effect of each dilution on the cell activation to the anti-IgE was calculated, for each experiment, according to the formula: Open in a separate window.

Discussion Human basophils are peripheral granulocytes involved in immune and inflammatory processes such as hypersensitivity and allergic reactions.

Cells of the immune system and inflammation. Evid Based Complement Alternat Med. Experimental studies on animal models. Human basophil degranulation triggered by very dilute antiserum against IgE. Mechanical agitation of very dilute antiserum against IgE has no effect on basophil staining properties.

Human basophil degranulation is not triggered by very dilute antiserum against human IgE. Enzymes may use several of these mechanisms simultaneously. For example, proteases such as trypsin perform covalent catalysis using a catalytic triad , stabilise charge build-up on the transition states using an oxyanion hole , complete hydrolysis using an oriented water substrate.

Enzymes are not rigid, static structures; instead they have complex internal dynamic motions — that is, movements of parts of the enzyme's structure such as individual amino acid residues, groups of residues forming a protein loop or unit of secondary structure , or even an entire protein domain.

These motions give rise to a conformational ensemble of slightly different structures that interconvert with one another at equilibrium. Different states within this ensemble may be associated with different aspects of an enzyme's function. For example, different conformations of the enzyme dihydrofolate reductase are associated with the substrate binding, catalysis, cofactor release, and product release steps of the catalytic cycle.

Allosteric sites are pockets on the enzyme, distinct from the active site, that bind to molecules in the cellular environment. These molecules then cause a change in the conformation or dynamics of the enzyme that is transduced to the active site and thus affects the reaction rate of the enzyme.

Allosteric interactions with metabolites upstream or downstream in an enzyme's metabolic pathway cause feedback regulation, altering the activity of the enzyme according to the flux through the rest of the pathway.

Some enzymes do not need additional components to show full activity. Others require non-protein molecules called cofactors to be bound for activity. These cofactors serve many purposes; for instance, metal ions can help in stabilizing nucleophilic species within the active site. Organic prosthetic groups can be covalently bound e. An example of an enzyme that contains a cofactor is carbonic anhydrase , which is shown in the ribbon diagram above with a zinc cofactor bound as part of its active site.

Enzymes that require a cofactor but do not have one bound are called apoenzymes or apoproteins. An enzyme together with the cofactor s required for activity is called a holoenzyme or haloenzyme. The term holoenzyme can also be applied to enzymes that contain multiple protein subunits, such as the DNA polymerases ; here the holoenzyme is the complete complex containing all the subunits needed for activity.

Coenzymes are small organic molecules that can be loosely or tightly bound to an enzyme. Coenzymes transport chemical groups from one enzyme to another. These coenzymes cannot be synthesized by the body de novo and closely related compounds vitamins must be acquired from the diet.

The chemical groups carried include:. Since coenzymes are chemically changed as a consequence of enzyme action, it is useful to consider coenzymes to be a special class of substrates, or second substrates, which are common to many different enzymes.

For example, about enzymes are known to use the coenzyme NADH. Coenzymes are usually continuously regenerated and their concentrations maintained at a steady level inside the cell. For example, NADPH is regenerated through the pentose phosphate pathway and S -adenosylmethionine by methionine adenosyltransferase. This continuous regeneration means that small amounts of coenzymes can be used very intensively.

For example, the human body turns over its own weight in ATP each day. As with all catalysts, enzymes do not alter the position of the chemical equilibrium of the reaction. In the presence of an enzyme, the reaction runs in the same direction as it would without the enzyme, just more quickly.

The rate of a reaction is dependent on the activation energy needed to form the transition state which then decays into products. Enzymes increase reaction rates by lowering the energy of the transition state. First, binding forms a low energy enzyme-substrate complex ES. Finally the enzyme-product complex EP dissociates to release the products. Enzymes can couple two or more reactions, so that a thermodynamically favorable reaction can be used to "drive" a thermodynamically unfavourable one so that the combined energy of the products is lower than the substrates.

For example, the hydrolysis of ATP is often used to drive other chemical reactions. Enzyme kinetics is the investigation of how enzymes bind substrates and turn them into products. In Leonor Michaelis and Maud Leonora Menten proposed a quantitative theory of enzyme kinetics, which is referred to as Michaelis—Menten kinetics. In the first, the substrate binds reversibly to the enzyme, forming the enzyme-substrate complex. This is sometimes called the Michaelis-Menten complex in their honor.

The enzyme then catalyzes the chemical step in the reaction and releases the product. This work was further developed by G. Haldane , who derived kinetic equations that are still widely used today. Enzyme rates depend on solution conditions and substrate concentration.

To find the maximum speed of an enzymatic reaction, the substrate concentration is increased until a constant rate of product formation is seen. This is shown in the saturation curve on the right.

Saturation happens because, as substrate concentration increases, more and more of the free enzyme is converted into the substrate-bound ES complex. At the maximum reaction rate V max of the enzyme, all the enzyme active sites are bound to substrate, and the amount of ES complex is the same as the total amount of enzyme.

V max is only one of several important kinetic parameters. The amount of substrate needed to achieve a given rate of reaction is also important.

This is given by the Michaelis-Menten constant K m , which is the substrate concentration required for an enzyme to reach one-half its maximum reaction rate; generally, each enzyme has a characteristic K M for a given substrate. Another useful constant is k cat , also called the turnover number , which is the number of substrate molecules handled by one active site per second. This is also called the specificity constant and incorporates the rate constants for all steps in the reaction up to and including the first irreversible step.

Because the specificity constant reflects both affinity and catalytic ability, it is useful for comparing different enzymes against each other, or the same enzyme with different substrates. At this point every collision of the enzyme with its substrate will result in catalysis, and the rate of product formation is not limited by the reaction rate but by the diffusion rate.

Enzymes with this property are called catalytically perfect or kinetically perfect. Michaelis—Menten kinetics relies on the law of mass action , which is derived from the assumptions of free diffusion and thermodynamically driven random collision.

Many biochemical or cellular processes deviate significantly from these conditions, because of macromolecular crowding and constrained molecular movement. Enzyme reaction rates can be decreased by various types of enzyme inhibitors. A competitive inhibitor and substrate cannot bind to the enzyme at the same time.

For example, the drug methotrexate is a competitive inhibitor of the enzyme dihydrofolate reductase , which catalyzes the reduction of dihydrofolate to tetrahydrofolate. This type of inhibition can be overcome with high substrate concentration. In some cases, the inhibitor can bind to a site other than the binding-site of the usual substrate and exert an allosteric effect to change the shape of the usual binding-site.

A non-competitive inhibitor binds to a site other than where the substrate binds. The substrate still binds with its usual affinity and hence K m remains the same.

However the inhibitor reduces the catalytic efficiency of the enzyme so that V max is reduced. In contrast to competitive inhibition, non-competitive inhibition cannot be overcome with high substrate concentration. An uncompetitive inhibitor cannot bind to the free enzyme, only to the enzyme-substrate complex; hence, these types of inhibitors are most effective at high substrate concentration.

In the presence of the inhibitor, the enzyme-substrate complex is inactive. A mixed inhibitor binds to an allosteric site and the binding of the substrate and the inhibitor affect each other. The enzyme's function is reduced but not eliminated when bound to the inhibitor. This type of inhibitor does not follow the Michaelis-Menten equation. An irreversible inhibitor permanently inactivates the enzyme, usually by forming a covalent bond to the protein.

In many organisms, inhibitors may act as part of a feedback mechanism. If an enzyme produces too much of one substance in the organism, that substance may act as an inhibitor for the enzyme at the beginning of the pathway that produces it, causing production of the substance to slow down or stop when there is sufficient amount.

This is a form of negative feedback. Major metabolic pathways such as the citric acid cycle make use of this mechanism. Since inhibitors modulate the function of enzymes they are often used as drugs. Many such drugs are reversible competitive inhibitors that resemble the enzyme's native substrate, similar to methotrexate above; other well-known examples include statins used to treat high cholesterol , [76] and protease inhibitors used to treat retroviral infections such as HIV.

For example, the poison cyanide is an irreversible enzyme inhibitor that combines with the copper and iron in the active site of the enzyme cytochrome c oxidase and blocks cellular respiration. Enzymes serve a wide variety of functions inside living organisms.

They are indispensable for signal transduction and cell regulation, often via kinases and phosphatases. Enzymes are also involved in more exotic functions, such as luciferase generating light in fireflies. An important function of enzymes is in the digestive systems of animals. Enzymes such as amylases and proteases break down large molecules starch or proteins , respectively into smaller ones, so they can be absorbed by the intestines.

Starch molecules, for example, are too large to be absorbed from the intestine, but enzymes hydrolyze the starch chains into smaller molecules such as maltose and eventually glucose , which can then be absorbed. Different enzymes digest different food substances. In ruminants , which have herbivorous diets, microorganisms in the gut produce another enzyme, cellulase , to break down the cellulose cell walls of plant fiber.

Several enzymes can work together in a specific order, creating metabolic pathways. After the catalytic reaction, the product is then passed on to another enzyme. Sometimes more than one enzyme can catalyze the same reaction in parallel; this can allow more complex regulation: Enzymes determine what steps occur in these pathways. Without enzymes, metabolism would neither progress through the same steps and could not be regulated to serve the needs of the cell.

Most central metabolic pathways are regulated at a few key steps, typically through enzymes whose activity involves the hydrolysis of ATP. Because this reaction releases so much energy, other reactions that are thermodynamically unfavorable can be coupled to ATP hydrolysis, driving the overall series of linked metabolic reactions. There are five main ways that enzyme activity is controlled in the cell. Enzymes can be either activated or inhibited by other molecules. For example, the end product s of a metabolic pathway are often inhibitors for one of the first enzymes of the pathway usually the first irreversible step, called committed step , thus regulating the amount of end product made by the pathways.

Such a regulatory mechanism is called a negative feedback mechanism , because the amount of the end product produced is regulated by its own concentration. This helps with effective allocations of materials and energy economy, and it prevents the excess manufacture of end products. Like other homeostatic devices , the control of enzymatic action helps to maintain a stable internal environment in living organisms.

Examples of post-translational modification include phosphorylation , myristoylation and glycosylation. Chymotrypsin , a digestive protease , is produced in inactive form as chymotrypsinogen in the pancreas and transported in this form to the stomach where it is activated. This stops the enzyme from digesting the pancreas or other tissues before it enters the gut.

This type of inactive precursor to an enzyme is known as a zymogen [85]: Enzyme production transcription and translation of enzyme genes can be enhanced or diminished by a cell in response to changes in the cell's environment. This form of gene regulation is called enzyme induction. For example, bacteria may become resistant to antibiotics such as penicillin because enzymes called beta-lactamases are induced that hydrolyse the crucial beta-lactam ring within the penicillin molecule.

Induction or inhibition of these enzymes can cause drug interactions. Enzymes can be compartmentalized, with different metabolic pathways occurring in different cellular compartments.

In multicellular eukaryotes , cells in different organs and tissues have different patterns of gene expression and therefore have different sets of enzymes known as isozymes available for metabolic reactions. This provides a mechanism for regulating the overall metabolism of the organism. For example, hexokinase , the first enzyme in the glycolysis pathway, has a specialized form called glucokinase expressed in the liver and pancreas that has a lower affinity for glucose yet is more sensitive to glucose concentration.

Since the tight control of enzyme activity is essential for homeostasis , any malfunction mutation, overproduction, underproduction or deletion of a single critical enzyme can lead to a genetic disease. The malfunction of just one type of enzyme out of the thousands of types present in the human body can be fatal. An example of a fatal genetic disease due to enzyme insufficiency is Tay-Sachs disease , in which patients lack the enzyme hexosaminidase. One example of enzyme deficiency is the most common type of phenylketonuria.

Many different single amino acid mutations in the enzyme phenylalanine hydroxylase , which catalyzes the first step in the degradation of phenylalanine , result in build-up of phenylalanine and related products. Some mutations are in the active site, directly disrupting binding and catalysis, but many are far from the active site and reduce activity by destabilising the protein structure, or affecting correct oligomerisation.

Another way enzyme malfunctions can cause disease comes from germline mutations in genes coding for DNA repair enzymes. Defects in these enzymes cause cancer because cells are less able to repair mutations in their genomes. This causes a slow accumulation of mutations and results in the development of cancers.

An example of such a hereditary cancer syndrome is xeroderma pigmentosum , which causes the development of skin cancers in response to even minimal exposure to ultraviolet light.

Enzymes are used in the chemical industry and other industrial applications when extremely specific catalysts are required. Enzymes in general are limited in the number of reactions they have evolved to catalyze and also by their lack of stability in organic solvents and at high temperatures.

As a consequence, protein engineering is an active area of research and involves attempts to create new enzymes with novel properties, either through rational design or in vitro evolution. From Wikipedia, the free encyclopedia. For the use of natural catalysts in organic chemistry, see Biocatalysis. Enzyme catalysis and Transition state theory.

Activation energy , Thermodynamic equilibrium , and Chemical equilibrium. A chemical reaction mechanism with or without enzyme catalysis. The enzyme E binds substrate S to produce product P. Saturation curve for an enzyme reaction showing the relation between the substrate concentration and reaction rate. An enzyme binding site that would normally bind substrate can alternatively bind a competitive inhibitor , preventing substrate access.

Dihydrofolate reductase is inhibited by methotrexate which prevents binding of its substrate, folic acid. The coenzyme folic acid left and the anti-cancer drug methotrexate right are very similar in structure differences show in green. As a result, methotrexate is a competitive inhibitor of many enzymes that use folates. Biology portal Molecular and cellular biology portal Metabolism portal Food portal. Histoire de l'academie royale des sciences.

A History of Science: Modern Development of the Chemical and Biological Sciences. Annales de chimie et de physique. Verhandlungen des naturhistorisch-medicinischen Vereins zu Heidelberg. Relevant passage on page In order to obviate misunderstandings and avoid cumbersome periphrases, [the author, a university lecturer] suggests designating as "enzymes" the unformed or not organized ferments, whose action can occur without the presence of organisms and outside of the same.

Retrieved 23 February Diastases, toxines et venins [ Microbiology Treatise: See Chapter 1, especially page 9. Problems and methods in enzyme research".

Journal of the Chemical Society Resumed: The Nobel Prize in Chemistry ". Archived from the original on 17 March Retrieved 6 March