壳聚糖抑菌机制的初步研究

壳聚糖在医学、食品、环保、日化用品等领域有着广泛而重要的应用.近年来,壳聚糖由于对不同的菌类都具有良好的抑菌效果而被研究者们密切关注.然而,有关壳聚糖抑菌机制的研究却并不多,其抑菌机制也没有被完全阐明.在本研究中,我们发现很多金属离子可以对壳聚糖的抑菌效果产生影响,高浓度金属离子(0.5%)可以使壳聚糖完全丧失抑菌活性.还发现金黄色葡萄球菌和白色念珠菌在壳聚糖的作用下会发生钾离子和ATP的渗漏,而且五万分子量的壳聚糖引起钾离子和ATP的渗漏大约比五千分子量壳聚糖多2到4倍.不同分子量的壳聚糖对金黄色葡萄球菌和白色念珠菌都具有较好的抑菌效果,但是引起钾离子和ATP的渗漏量却存在很大差异,这说明小分子量壳聚糖很可能存在与大分子量壳聚糖不同的抑菌机制.     

Influence of alkali metal cations on the transport of calcium by phosphatidate in multi-phase systems

The effects of alkali metal cations on the rates at which Ca2+ and phosphatidic acid were cotransported from aqueous to hydrocarbon medium were examined. The alkali metal cations remained in the aqueous phase yet specifically influenced the transport of Ca2+ into the hydrocarbon solvent. For the physiological cations, Na+ and K+, there were critical concentration ranges in which small changes in concentration effected sharp changes in transport rates. The maximal rate observed with Na+ was an order of magnitude greater than that with K+; however, unlike Na+, K+ promoted low levels of transport below the critical concentration range. Li+ effected only low levels of transport even at high concentrations, whereas Rb+ and Cs+ induced transport at rates proportional to their concentrations. These results are discussed in terms of a classical ionophore model for the complex composed of a neutral phosphatidic acid dimer bridged by Ca2+.     

Effect of various components of the fungal cell wall on adhesion of Candida albicans to the mouth mucosa epithelium in vitro]

An influence of mannan++, its component methyl-D-mannopyranoside+ and N-acetylglucosamine on in vitro adhesion of Candida albicans strains to buccal mucosal epithelium was studied. These substances inhibited adhesion when added to adherence test in a concentration of 10 mg/ml and 25 mg/ml despite whether were added to the test incubation medium or when preincubated with fungi or epithelial cells. Preincubation of fungal cells and epithelial cells with mannan had no influence on attachment; preincubation of epithelial cells with methyl-D-mannopyranoside+ and N-acetylglucosamine decreased adherence significantly. On the other hand preincubation of fungal calls with methyl-D-mannopyranoside+ increased their adhesive properties, having no influence on adherence after preincubation of fungi with N-acetylglucosamine.     

Effect of intracellular Na+ on platelet aggregation and Ca2+ mobilization

The effects of ionophores, which can carry alkali metal cations, on platelet aggregation were examined. At an alkaline extracellular pH, alkali metal cation/H+ exchanger nigericin accelerated aggregation in K+-enriched medium, whereas it rather inhibited aggregation in Na+-enriched medium, even though the intracellular pH was only slightly alkaline. The inhibitory effect of Na+ on platelet aggregation was more clearly shown with the alkali metal cation exchanger gramicidin D. The ionophore had no effect or a slightly accelerative effect on aggregation in K+-enriched medium, whereas it significantly inhibited aggregation induced by thrombin, ADP and platelet activating factor in Na+-enriched medium. Fluorescence studies on fura-2-labeled platelets revealed that in Na+-enriched medium gramicidin D inhibited agonist-induced Ca2+ mobilization both in the presence and absence of extracellular Ca2+. These results suggest that the intracellular Na+ inhibits platelet aggregation by inhibiting Ca2+ mobilization.     

Roles of sodium and potassium ions on p-aminohippurate transport in rabbit kidney slices.

This investigation was principally undertaken to test the ionic gradient hypothesis as applied to active p-aminohippurate uptake in the rabbit kidney cortical slice preparation. Efflux of p-aminohippurate from the slice was shown to be independent of external Na+ concentration. Transferring slices from a low sodium preincubation to a high sodium incubation medium containing p-aminohippurate increased intracellular concentrations of both Na+ and K+, and p-aminohippurate accumulation occurred. Transferring slices from a low sodium preincubation to a high sodium incubation medium containing ouabain and p-aminohippurate resulted in a net increase in intracellular Na+ concentration but no p-aminohippurate accumulation occurred. Different combinations of preincubation and incubation media gave a high to low array of intracellular Na+ concentrations and these directly reflected their respective p-aminohippurate uptake. These results suggest that the Na+ gradient hypothesis does not adequately explain the transport of organic acids in rabbit kidney. These results also suggest that Na+ possibly has an intracellular role through its stimulation of (Na+ + K+)-ATPase channeled to energizing the p-aminohippurate accumulative mechanism.     

铯进入红细胞的NMR研究

在生理及生化研究中铯离子的行为受到极大的关注,因为可由它来阐述碱金属离子输运及酶活性等一些基本功能。当所研究的体系中没有K~+时,Cs离子有类似K~+的功能,它能激活Na/K ATPase。Cs进入细胞的速率及激活Na/K ATPase所要求的浓度与被研究系统的条件密切有关。本文用核磁共振谱方法测定红细胞内外的Cs~+及Cs~+进入细胞的速率。结果表明Cs-133的核磁共振谱是研究生物体系离子的分布及输运的极好工具。     

The antagonistic effect of Saccharomyces boulardii on Candida albicans filamentation, adhesion and biofilm formation

The dimorphic fungus Candida albicans is a member of the normal flora residing in the intestinal tract of humans. In spite of this, under certain conditions it can induce both superficial and serious systemic diseases, as well as be the cause of gastrointestinal infections. Saccharomyces boulardii is a yeast strain that has been shown to have applications in the prevention and treatment of intestinal infections caused by bacterial pathogens. The purpose of this study was to determine whether S. boulardii affects the virulence factors of C. albicans . We demonstrate the inhibitory effect of live S. boulardii cells on the filamentation (hyphae and pseudohyphae formation) of C. albicans SC5314 strain proportional to the amount of S. boulardii added. An extract from S. boulardii culture has a similar effect. Live S. boulardii and the extract from S. boulardii culture filtrate diminish C. albicans adhesion to and subsequent biofilm formation on polystyrene surfaces under both aerobic and microaerophilic conditions. This effect is very strong and requires lower doses of S. boulardii cells or concentrations of the extract than serum-induced filamentation tests. Saccharomyces boulardii has a strong negative effect on very important virulence factors of C. albicans , i.e. the ability to form filaments and to adhere and form biofilms on plastic surfaces.     

Dependence of the intracellular concentrations of univalent ions and hydrogenase activity on the salt composition and pH of the medium in the haloalkaliphilic sulfate-reducing bacterium Desulfonatronum thiodismutans

It has been shown that the intracellular concentrations of Na+, K+, and Cl- ions in Desulfonatronum thiodismutans depend on the extracellular concentration of Na' ions. An increase in the extracellular concentration of Na+ results in the accumulation of K+ ions in cells, which points to the possibility that these ions perform an osmoprotective function. When the concentration of the NaCI added to the medium was increased to 4%, the concentration gradient of Cl- ions changed insignificantly. It was found that D. thiodismutans contains two forms of hydrogenase--periplasmic and cytoplasmic. Both enzymes are capable of functioning in solutions with high ionic force; however they exhibit different sensitivities to Na+, K+, and Li+ salts and pH. The enzymes were found to be resistant to high concentrations of Na+ and K+ chlorides and Na+ bicarbonate. The cytoplasmic hydrogenase differed significantly from the periplasmic one in having much higher salt tolerance and lower pH optimum. The activity of these enzymes depended on the nature of both the cationic and anionic components of the salts. For instance, the inhibitory effect of NaCl was less pronounced than that of LiCl, whereas Na+ and Li+ sulfates inhibited the activity of both hydrogenase types to an equal degree. The highest activity of these enzymes was observed at low Na+ concentrations, close to those typical of cells growing at optimal salt concentrations.     

The modifications of the final stages of the complement reaction by alkali metal cations.

We studied the effects of alkali metal cations on the terminal stages of complement lysis of human and sheep HK erythrocytes. Sensitized erythrocytes (EA) were reacted with limited amounts of complement for 1 hr at 37 degrees C in buffer containing 147 mM NaCl (Na buffer), which resulted in 10-40% lysis. The unlysed cells were washed with Na buffer at 0-2 degrees C and incubated for 1 hr at 37 degrees C in buffers containing 147 mM of the various alkali metal cations. Although additional lysis (25 to 65%) occurred with K, Rb, or Cs buffer, only minor degrees developed with Na or Li buffer, only minor degrees developed with Na or Li buffer. Intermediate levels occurred with 100 mM of the divalent alkali cations. Halogen ions and SCN-(147 MM), Ca++ (0.15mM), and Mg++ (0.5 mM) did not alter the effect of the alkali metal cations. Lysis occurring in K+, Rb+ or Cs+ proceeded without lag, was temperature dependent with an optimum of 43 degrees C, and had a pH optimum of 6.5. Lysis in K and Na buffers was unaffected by 10(-3) to 10(-5) M ouabain. Experiments with mixtures of cations indicated that Na+ had a mild inhibitory effect that could be totally overcome by K+, partially by Rb+, and not at all by Cs+. Li+ had a strong inhibitory effect, 6 X 10(-5) M causing 50% inhibition in buffers containing 147 mM K+, Rb+, or Cs+. By using intermediate complexes of EA and purified complement components we demonstrated that K+ enhances the lytic action of C8 on EAC1-7 as well as that of C9 on EAC1-8. It was known that Li+ facilitates lysis when acting on the entire complement reaction. We found that Li+ enhanced the lytic action of C8 on EAC1-7, with a kinetic that differed from that of the K+ effect. In addition, Li+ inhibited the enhancing effect of K+ upon lysis of EAC1-8 by C9. This occurred at concentration of Li+ similar to those which inhibited the additional lysis by K+, Rb+, and Cs+ of cells that were pretreated in Na buffer with the entire complement sequence. We propose that the major effects of alkali metal cations on complement lysis are due to their interaction with C8 and/or membrane constitutes.     

The influence of potassium ion (K+) on digoxin-induced inhibition of porcine cerebral cortex Na+ / K+-ATPase

The in vitro influence of potassium ion modulations, in the concentration range 2 mM-500 mM, on digoxin-induced inhibition of porcine cerebral cortex Na+ / K+-ATPase activity was studied. The response of enzymatic activity in the presence of various K+ concentrations to digoxin was biphasic, thereby, indicating the existence of two Na+ / K+-ATPase isoforms, differing in the affinity towards the tested drug. Both isoforms showed higher sensitivity to digoxin in the presence of K+ ions below 20 mM in the medium assay. The IC50 values for high/low isoforms 2.77 x 10(-6) M / 8.56 x 10(-5) M and 7.06 x 10(-7) M / 1.87 x 10(-5) M were obtained in the presence of optimal (20 mM) and 2 mM K+, respectively. However, preincubation in the presence of elevated K+ concentration (50-500 mM) in the medium assay prior to Na+ / K+-ATPase exposure to digoxin did not prevent the inhibition, i.e. IC50 values for both isoforms was the same as in the presence of the optimal K+ concentration. On the contrary, addition of 200 mM K+ into the medium assay after 10 minutes exposure of Na+ / K+-ATPase to digoxin, showed a time-dependent recovery effect on the inhibited enzymatic activity. Kinetic analysis showed that digoxin inhibited Na+ / K+-ATPase by reducing maximum enzymatic velocity (Vmax) and Km, implying an uncompetitive mode of interaction.     

Synthesis, DNA cleavage, and cytotoxicity of a series of bis(propargylic) sulfone crown ethers

Compounds that couple molecular recognition of specific alkali metal ions with DNA damage may display selective cleavage of DNA under conditions of elevated alkali metal ion levels reported to exist in certain cancer cells. We have prepared a homologous series of compounds in which a DNA reactive moiety, a bis(propargylic) sulfone, is incorporated into an alkali metal ion binding crown ether ring. Using the alkali metal ion pricrate extraction assay, the ability of these crown ethers to bind Li(+), Na(+), and K(+) ions was determined. For the series of crown ethers, the association constants for Li(+) ions are generally low (< 2 x 10(4)M(-1)). Only two of the bis(propargylic) sulfone crown ethers associate with Na(+) or K(+) ions (K(a) 4-8 x 10(4)M(-1)), with little discrimination between Na(+) or K(+) ions. The ability of these compounds to cleave supercoiled DNA at pH 7.4 in the presence of Li(+), Na(+), and K(+) ions was determined. The two crown ethers that bind Na(+) and K(+) display a modest increase in DNA cleavage efficiency in the presence of Na(+) or K(+) ions as compared to Li(+) ions. These two bis(propargylic) sulfone crown ethers are also more cytotoxic against a panel of human cancer cell lines when compared to a non-crown ether macrocyclic bis(propargylic) sulfone.     

Effects of L-phenylalanine on acetylcholinesterase, (Na+,K+)-ATPase and Mg2+-ATPase activities in adult rat whole brain and frontal cortex

The effect of different L-phenylalanine (Phe) concentrations (0.12-12.1 mM) on acetylcholinesterase (AChE), (Na+,K+)-ATPase and Mg2+-ATPase activities was investigated in homogenates of adult rat whole brain and frontal cortex at 37 degrees C. AChE, (Na+,K+)-ATPase and Mg2+-ATPase activities were determined after preincubation with Phe. AChE activity in both tissues showed a decrease up to 18% (p<0.01) with Phe. Whole brain Na+,K+-ATPase was stimulated by 30-35% (p<0.01) with high Phe concentrations, while frontal cortex Na+,K+-ATPase was stimulated by 50-55% (p<0.001). Mg2+-ATPase activity was increased only in frontal cortex with high Phe concentrations. It is suggested that: a) The inhibitory effect of Phe on brain AChE is not influenced by developmental factors, while the stimulation of Phe on brain Na+,K+-ATPase is indeed affected; b) The stimulatory effect of Phe on rat whole brain Na+,K+-ATPase is decreased with age; c) Na+,K+-ATPase is selectively more stimulated by high Phe concentrations in frontal cortex than in whole brain homogenate; d) High (toxic) Phe concentrations can affect Mg2+-ATPase activity in frontal cortex, but not in whole brain, thus modulating the amount of intracellular Mg2+.     

Right-handed and left-handed helices of poly(dA-dC) X (dG-dT).

The secondary structures of poly(dA-dC) X (dG-dT) were studied using CD and IR spectroscopies. We give spectroscopic evidence of secondary structure transitions of poly(dA-dC) X (dG-dT) from a B to a Z-like helix, induced by transition metal ions (Ni2+) in presence of high concentrations of Cs+ and Na+. In the presence of Na+, the B in equilibrium Z transition occurs at any temperature, whereas premelting conditions are required in presence of Cs+. For these two alkali ions the Z-like form is only induced by Ni2+ ions through their specific interactions at N7 of purines, under conditions of low water activity due to the high alkali salt concentration. We also show that the CD spectrum obtained in presence of Cs+ ions and characterized by a negative band at 275 nm, cannot be interpreted in terms of Z-like left-handed helix but reflects a modified B right-handed helix.     

Effects of pH on the interaction of ligands with the (H+ + K+)-ATPase purified from pig gastric mucosa

The effects of K+, Na+ and ATP on the gastric (H+ + K+)-ATPase were investigated at various pH. The enzyme was phosphorylated by ATP with a pseudo-first-order rate constant of 3650 min-1 at pH 7.4. This rate constant increased to a maximal value of about 7900 min-1 when pH was decreased to 6.0. Alkalinization decreased the rate constant. At pH 8.0 it was 1290 min-1. Additions of 5 mM K+ or Na+, did not change the rate constant at acidic pH, while at neutral or alkaline pH a decrease was observed. Dephosphorylation of phosphoenzyme in lyophilized vesicles was dependent on K+, but not on Na+. Alkaline pH increased the rate of dephosphorylation. K+ stimulated the ATPase and p-nitrophenylphosphatase activities. At high concentrations K+ was inhibitory. Below pH 7.0 Na+ had little or no effect on the ATPase and p-nitrophenylphosphatase, while at alkaline pH, Na+ inhibited both activities. The effect of extravesicular pH on transport of H+ was investigated. At pH 6.5 the apparent Km for ATP was 2.7 microM and increased little when K+ was added extravesicularly. At pH 7.5, millimolar concentrations of K+ increased the apparent Km for ATP. Extravesicular K+ and Na+ inhibited the transport of H+. The inhibition was strongest at alkaline pH and only slight at neutral or acidic pH, suggesting a competition between the alkali metal ions and hydrogen ions at a common binding site on the cytoplasmic side of the membrane. Two H+-producing reactions as possible candidates as physiological regulators of (H+ + K+)-ATPase were investigated. Firstly, the hydrolysis of ATP per se, and secondly, the hydration of CO2 and the subsequent formation of H+ and HCO3-. The amount of hydrogen ions formed in the ATPase reaction was highest at alkaline pH. The H+/ATP ratio was about 1 at pH 8.0. When CO2 was added to the reaction medium there was no change in the rate of hydrogen ion transport at pH 7.0, but at pH 8.0 the rate increased 4-times upon the addition of 0.4 mM CO2. The results indicate a possible co-operation in the production of acid between the H+ + K+-ATPase and a carbonic anhydrase associated with the vesicular membrane.     

Effects of sodium and potassium ions on the elementary steps in the reaction of Na+-K+-dependent ATPase1.

The effects of Na+ and K+ ions on the elementary steps in the reaction of Na+-K+-dependent ATPase (EC 3.6.1.3) were investigated in 0.5-600mM NaCL and 0-10mM KCL, at a fixed concentration (1mM) OF MgCL2, AT PH 8.5 and at 15 degrees. The data were analyzed on the basis of the reaction mechanism in which a phosphorylated intermediate, E ADP P (abbreviated as EP), is formed via two kinds of enzyme-substrate comples, E1ATP and E2ATP, and EP is in equilibrium with E2ATP, and is hydrolyzed to produce P1 and ADP. The following results were obtained: 1. The rate od E2ATP-formation, vf, increased with increase in the Na+ concentration, reached a maximum level, and then decreased with further increase in the Na+ concentration at various K+ concentrations. The value of vf was given as (see article). 2. The reciprocal of the equilibrium constants, K2, of the step E1ATPEQUILIBRIUM E ADP P in the presence of low concentrations of Na+ was larger than that in the presence of high concrntrations of Na+, indicating that the equilibrium shifted markedly toward E2ATP at low concentrations of Na+. The relation of K3 with Na concentration was rather complicated on varying the concentration of K+. However, generally speaking, it increased with increase in the K+ concentration. 3. The decomposition of EP was markedly activated by even low concentrations of K+, and inhibited by high concentrations of Na+. The inhibition by Na+ was partially suppressed by K+. The rate constant of EP-decomposition, vo/(EP), was given by (see article) where (vo/(EP) K+EQUALS0 was the value of vo/[EP] in the absence of K+.     

Adherence and biofilm formation of non-Candida albicans Candida species

Most cases of candidosis have been attributed to Candida albicans, but recently non-C. albicans Candida species have been identified as frequent human pathogens. Candida pathogenicity has been attributed to several factors, including adhesion to medical devices and/or host cells, biofilm formation, and secretion of hydrolytic enzymes (proteases, phospholipases and haemolysins). Although 'new'Candida species are emerging, there is still a lack of information about their pathogenicity. This review discusses recent advances in our knowledge of Candida glabrata, Candida parapsilosis and Candida tropicalis virulence factors, specifically those of adhesion and biofilm formation, which are key components in Candida pathogenicity.     

The enhanced ATPase activity of glutathione-substituted actin provides a quantitative approach to filament stabilization

[Cys374]glutathionyl-actin was prepared by isolation of the reaction product of G-actin with Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid], followed by reaction with glutathione. Filaments of this actin disulfide are susceptible to even weak shearing stress as exerted, for example, by heating to 37 degrees C. This treatment produces a 25-fold enhanced steady-state ATPase activity as compared to unsubstituted F-actin at room temperature. Monitoring the reduction of this enhanced ATPase activity is a reliable method for quantifying the effectiveness of filament-stabilizing agents and for determining their apparent dissociation constants. A detailed comparative study of filament-stabilizing agents was performed, and some hitherto unknown filament-protecting effects were revealed. Inorganic phosphate provides stabilization only to a maximum of 45% ATPase inhibition, but reaches this effect already at cytoplasmic Pi concentrations (approximately 4 mM). Arsenate seems to bind with similar affinity, but with distinctly less protective activity (maximum of 16%). High concentrations of alkali ions provide a more effective protection (maximum of 95%), Li+ being more efficient than Na+ and K+. Divalent cations (Ca2+, Mg2+) had a strong stabilizing effect on KCl-polymerized actin; we confirmed the presence of two distinct classes of binding sites for divalent metal ions with moderate and low affinities, apparent in a strong stabilizing effect on KCl-polymerized actin. The stabilizing effects of KCl and Pi are independent and additive. Correspondingly, at K2HPO4 concentrations greater than 4 mM, K+ ions contribute considerably to stabilization. In the presence of 100 mM KCl plus 4 mM Pi, conditions which mimic the physiological environment, filament protection is nearly as effective as with the mushroom toxin phalloidin. The strong stabilizing effect of phalloidin occurred at concentrations far below stoichiometric, suggesting a very high degree of cooperativity in its interaction with actin filaments.     

Ammonium inhibition of Arabidopsis root growth can be reversed by potassium and by auxin resistance mutations aux1, axr1, and axr2.

A novel effect of ammonium ions on root growth was investigated to understand how environmental signals affect organ development. Ammonium ions (3-12 mM) were found to dramatically inhibit Arabidopsis thaliana seedling root growth in the absence of potassium even if nitrate was present. This inhibition could be reversed by including in the growth medium low levels (20-100 microM) of potassium or alkali ions Rb+ and Cs+ but not alkali ions Na+ and Li+. The protective effect of low concentrations of potassium is not due to an inhibition of ammonium uptake. Ammonium inhibition is reversible, because root growth was restored in ammonium-treated seedlings if they were subsequently transferred to medium containing potassium. It is known that plant hormones can inhibit root growth. We found that mutants of Arabidopsis resistant to high levels of auxin and other hormones (aux1, axr1, and axr2) are also resistant to the ammonium inhibition and produce roots in the absence of potassium. Thus, the mechanisms that mediate the ammonium inhibition of root development are linked to hormone metabolic or signaling pathways. These findings have important implications for understanding how environmental signals, especially mineral nutrients, affect plant root development.     

Tuning the voltage dependence of tetraethylammonium block with permeant ions in an inward-rectifier K+ channel.

To understand the role of permeating ions in determining blocking ion-induced rectification, we examined block of the ROMK1 inward-rectifier K+ channel by intracellular tetraethylammonium in the presence of various alkali metal ions in both the extra- and intracellular solutions. We found that the channel exhibits different degrees of rectification when different alkali metal ions (all at 100 mM) are present in the extra- and intracellular solution. A quantitative analysis shows that an external ion site in the ROMK1 pore binds various alkali metal ions (Na+, K+, Rb+, and Cs+) with different affinities, which can in turn be altered by the binding of different permeating ions at an internal site through a nonelectrostatic mechanism. Consequently, the external site is saturated to a different level under the various ionic conditions. Since rectification is determined by the movement of all energetically coupled ions in the transmembrane electrical field along the pore, different degrees of rectification are observed in various combinations of extra- and intracellular permeant ions. Furthermore, the external and internal ion-binding sites in the ROMK1 pore appear to have different ion selectivity: the external site selects strongly against the smaller Na+, but only modestly among the three larger ions, whereas the internal site interacts quite differently with the larger K+ and Rb+ ions.     

Inhibition of Candida adhesion to buccal epithelial cells by an aqueous extract of Allium sativum (garlic)

The effect of pre-incubation of either Candida or buccal epithelial cells (BEC) with different concentrations of aqueous garlic extract (AGE) was investigated, as well as the effect of mouth rinse with AGE on the adhesion of yeast to BEC. Adhesion of Candida spp. to BEC was significantly reduced after both short and long time exposure of yeast to AGE. A similar inhibition of adherence was observed upon preincubation of BEC with AGE. The adherence-inhibition activity of AGE treatment was antagonized by thiols such as L-cysteine, glutathione and 2-mercaptoethanol. In addition, germ-tube formation was suppressed when C. albicans cells were pretreated with AGE. There was a significant reduction in the adherence of yeasts to BEC collected immediately or 15 min after an oral rinse with AGE. No statistical significance in the adhesion of BEC collected 30 min after oral rinse with AGE and control BEC was observed. The diminished adherence of C. albicans to BEC after exposure to various concentrations of garlic may have clinical relevance.