Mechanism of slow-binding inhibition of human leukocyte elastase by trifluoromethyl ketones

Kinetics of inhibition have been determined for the interaction of human leukocyte elastase (HLE) with two series of peptide trifluoromethyl ketones (TFMKs): X-Val-CF3,X-Pro-Val-CF3,X-Val-Pro-Val-CF3, and X-Lys(Z)-Val-Pro-Val-CF3, where X is MeOSuc or Z. These compounds are "slow-binding" inhibitors of HLE and, thus, allow the determination of Ki, the dissociation constant for the stable complex of inhibitor and enzyme, as well as kon and koff, the rate constants for formation and decomposition of this complex. Maximal potency is reached with Z-Lys(Z)-Val-Pro-Val-CF3, which displays a Ki less than 0.1 nM. Upon binding to HLE, these compounds undergo addition by the hydroxyl of the active site serine to form a hemiketal. The evidence supporting a hemiketal intermediate includes Ki values of 1.6 and 80,000 nM for Z-Val-Pro-Val-CF3 and its alcohol analogue, linear free energy correlations between inhibitory potency and catalytic efficiency for structurally related TFMKs and substrates, and the pH dependence of kon for the inhibition of HLE by Z-Val-Pro-Val-CF3, which is sigmoidal and displays a pKa of 6.9. Hemiketal formation is probably not rate limiting, however. Kinetic solvent isotope effects of unity suggest that kon cannot be rate limited by a reaction step, like hemiketal formation, that is subject to protolytic catalysis. A general mechanism that is consistent with these results is one in which formation of the hemiketal is rapid and is followed or preceded by a slow step that rate limits kon.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of human serine proteases by substituted 2-azetidinones.

trans-4-Ethoxycarbonyl-3-ethyl-1-(4-nitrophenyl-sulfonyl)-azetidin -3-one described by Firestone et al. (1990, Tetrahedron 46, 2255) as an inhibitor of human leucocyte elastase (HLE) displayed potent, time-dependent inhibition of both HLE and human cathepsin G (Cat-G). The cis-isomer was 7- and 180-fold less active, respectively. The mechanism likely involves opening of the beta-lactam ring by the active site serine to form an acyl-enzyme intermediate(s). This intermediate partitions with ratios of 4:1 between turnover of the inhibitor and formation of relatively stable enzyme-inhibitor complexes from both enzymes. The final HLE-inhibitor complex reactivated with a half-life of 48 h at 25 degrees C and was 16-fold more stable than the Cat-G-inhibitor complex. The stability of the acyl-enzymes supports a "double hit" chemical mechanism involving both serine acylation and alkylation of the histidine. These observations suggest that beta-lactams may be developed as a class of serine protease inhibitors.     

Neutrophil serine proteinases activate human nonepithelial cells to produce inflammatory cytokines through protease-activated receptor 2

Protease-activated receptors (PARs) compose a family of G protein-coupled receptors activated by proteolysis with exposure of their tethered ligand. Recently, we reported that a neutrophil-derived serine proteinase, proteinase 3 (PR3), activated human oral epithelial cells through PAR-2. The present study examined whether other neutrophil serine proteinases, human leukocyte elastase (HLE), and cathepsin G (Cat G) activate nonepithelial cells, human gingival fibroblasts (HGF). HLE and Cat G as well as PR3 activated HGF to produce IL-8 and monocyte chemoattractant protein 1. Human oral epithelial cells but not HGF express mRNA and protein of secretory leukocyte protease inhibitor, an inhibitor of HLE and Cat G, and recombinant secretory leukocyte protease inhibitor clearly inhibited the activation of HGF induced by HLE and Cat G but not by PR3. HGF express PAR-1 and PAR-2 mRNA in the cells and the proteins on the cell surface. HLE and Cat G cleaved the peptide corresponding to the N terminus of PAR-2 with exposure of its tethered ligand. Treatment with trypsin, an agonist for PAR-2, and a synthetic PAR-2 agonist peptide induced intracellular Ca(2+) mobilization and rendered cells refractory to subsequent stimulation with HLE and Cat G. The production of cytokine induced by HLE and Cat G and the PAR-2 agonist peptide was completely abolished by inhibition of phospholipase C. These findings suggest that neutrophil serine proteinases have equal ability to activate human nonepithelial cells through PAR-2 to produce inflammatory cytokines and may control a number of inflammatory processes such as periodontitis.     

2,4,5-Triphenylisothiazol-3 (2H)-one 1,1-dioxides as inhibitors of human leukocyte elastase

A series of substituted 2,4,5-triphenylisothiazol-3(2H)-one 1,1-dioxides 9 was synthesized and investigated as inhibitors of human leukocyte elastase (HLE). All compounds were found to inhibit HLE in a time-dependent manner and most of them exhibited kobs/[I] values > 300M(-1)s(-1). The most potent 3-oxosultam of this series was 91 (kobs/[I] = 2440 M(-1)s(-1)). Kinetic investigations performed with 9g and different substrate concentrations did not allow to clearly distinguish between a competitive or noncompetitive mode of inhibition. A more complex interaction is supported by the failure of a linear dependency of kobs values on the inhibitor concentration.     

6-Acylamino-2-[(alkylsulfonyl)oxy]-1H-isoindole-1,3-dione mechanism-based inhibitors of human leukocyte elastase

A study of various 2-[(alkylsulfonyl)oxy]-6-substituted-1H-isoindole-1,3-diones' inhibition of chymotrypsin compared to inhibition of HLE reveals that acylamino substitution in the 6-position increases selectivity and potency of these inhibitors for HLE. The best HLE inhibitor in this series was 6-(methylglutaryl)amino-2-[(ethylsulfonyl)oxy]-1H-isoindole-1,3-di one with a kobs/[I] = 220,000 M(-1) s(-1).     

Potential of pyrazolooxadiazinone derivatives as serine protease inhibitors

As a part of an investigation on molecular hybrids as new serine protease inhibitors, the pyrazolo [4,3-c][1,2,5]oxadiazin-3(5H)-one ring system was selected as a model of potential mechanism-based inhibitors. Due to the inherent reactivity of this system an optimal balance between susceptibility to nucleophilic attack and stability in solvents was sought prior to development as therapeutic agents. Substitutions on N5 and C7 of the supporting pyrazole ring with either aliphatic or aromatic groups (compounds 2 a-m) and the replacement of the carbonyl oxygen on the reactive oxadiazinone ring with sulfur (compounds 3a,i) were explored. Two members (2i and 2k) of this class of inhibitors displayed time-dependent inhibition of HLE suggesting mechanism-based inhibition. The observation that HLE generated a product(s) from compound 2i which displayed an identical UV-Visible spectrum to that observed during non-enzymatic hydrolysis further supports this proposal. FlexX-based docking of these compounds into a model of the human leukocyte elastase (HLE) active site produced a molecular model of the inhibitor-enzyme interaction.     

Mechanism of inactivation of human leukocyte elastase by a chloromethyl ketone: kinetic and solvent isotope effect studies

The mechanism of inactivation of human leukocyte elastase (HLE) by the chloromethyl ketone MeOSuc-Ala-Ala-Pro-Val-CH2Cl was investigated. The dependence of the first-order rate constant for inactivation on concentration of chloromethyl ketone is hyperbolic and suggests formation of a reversible "Michaelis complex" prior to covalent interaction between the enzyme and inhibitor. However, the observed Ki value is 10 microM, at least 10-fold lower than dissociation constants for complexes formed from interaction of HLE with structurally related substrates or reversible inhibitors, and suggests that Ki is a complex kinetic constant, reflecting the formation and accumulation of both the Michaelis complex and a second complex. It is proposed that this second complex is a hemiketal formed from attack of the active site serine on the carbonyl carbon of the inhibitor. The accumulation of this intermediate may be a general feature of reactions of serine proteases and chloromethyl ketones derived from specific peptides and accounts for the very low Ki values observed for these reactions. The solvent deuterium isotope effect (SIE) on the inactivation step (ki) is 1.58 +/- 0.07 and is consistent with rate-limiting, general-catalyzed attack of the active site His on the methylene carbon of the inhibitor with displacement of chloride anion. The general catalyst is thought to be the active site Asp. In contrast, the SIE on the second-order rate constant for HLE inactivation, ki/Ki, is inverse and equals 0.64 +/- 0.05.(ABSTRACT TRUNCATED AT 250 WORDS)     

X-ray crystal structure of the complex of human leukocyte elastase (PMN elastase) and the third domain of the turkey ovomucoid inhibitor.

Orthorhombic crystals diffracting beyond 1.7 A resolution, have been grown from the stoichiometric complex formed between human leukocyte elastase (HLE) and the third domain of turkey ovomucoid inhibitor (OMTKY3). The crystal and molecular structure has been determined with the multiple isomorphous replacement technique. The complex has been modeled using the known structure of OMTKY3 and partial sequence information for HLE, and has been refined. The current crystallographic R-value is 0.21 for reflections from 25 to 1.8 A resolution. HLE shows the characteristic polypeptide fold of trypsin-like serine proteinases and consists of 218 amino acid residues. However, several loop segments, mainly arranged around the substrate binding site, have unique conformations. The largest deviations from the other vertebrate proteinases of known spatial structure are around Cys168. The specificity pocket is constricted by Val190, Val216 and Asp226 to preferentially accommodate medium sized hydrophobic amino acids at P1. Seven residues of the OMTKY3-binding segment are in specific contact with HLE. This interaction and geometry around the reactive site are similar as observed in other complexes. It is the first serine proteinase glycoprotein analysed, having two sugar chains attached to Asn159 and to residue 109.     

Inhibition of human leukocyte elastase by phosphate esters of N-hydroxysuccinimide and its derivatives: direct observation of a phosphorylated enzyme by 31P nuclear magnetic resonance spectroscopy

A series of phosphate esters derived from N-hydroxysuccinimide and 3-alkyl-N-hydroxysuccinimide have been synthesized and found to be potent time-dependent irreversible inhibitors of human leukocyte elastase (HLE). The observed inhibitory activity in this series of compounds correlated well with the known preference of HLE for substrates with small hydrophobic side chains. Maximum potency was reached when a favorable aromatic interaction involving a phenyl group present in the inhibitor and an aromatic residue located in the vicinity of the S2' subsite was operative. 31P NMR spectroscopy was used to probe the mechanism of action of these compounds. Direct evidence is presented in support of a mechanism involving phosphorylation of the active site serine. These compounds constitute a new class of hydrolytically stable phosphorylating agents.     

Structure of porcine pancreatic elastase complexed with FR901277, a novel macrocyclic inhibitor of elastases, at 1.6 A resolution

Human leukocyte elastase (HLE) is a serine protease that contributes to tissue destruction in various disease states-for example, in emphysema. FR901277 is a natural product isolated from the culture filtrate of Streptomyces resistomicificus and is a potent inhibitor of both HLE and porcine pancreatic elastase (PPE). FR901277 consists of four normal amino acids and three unusual amino acids, and is a unique bicyclic peptide compound. The crystal structure of PPE complexed with FR901277 has been determined at 1.6 A resolution. The Ogamma atom of Ser-195 in PPE did not form a covalent bond with FR901277, but formed a hydrogen bond with the Nvarepsilon atom of His-57. On the other hand, the portion from L-Orn(1) through dehydroxyThr(3) in FR901277 formed an antiparallel beta-sheet structure with the backbone of the active site in PPE. The S4 through S2' binding subsites in PPE were all occupied by the hydrophobic side chains of the inhibitor molecule. Especially, the ethylidene moiety of FR901277 occupied the S1 specific pocket, indicating a CH/pi interaction. In addition, the isopropyl side chain of L-Val(7) was located at the enzyme surface between the S2 and S1' pockets with several van der Waals contacts. However, the amino acid (4) residue was not involved in a significant interaction with PPE. Comparison of inhibitor structures in different environments showed that FR901277 has a highly rigid bicyclic framework; however, it can slightly change its conformation according to the circumstances. The binding mode of FR901277 at the active site of PPE was directly applicable to that in HLE, after consideration of induced fit. The structure of the PPE-FR901277 complex provided much information regarding potential sites for modification of the physicochemical properties of FR901277.     

Structure-activity studies of fluoroketone inhibitors of alpha-lytic protease and human leukocyte elastase

We have synthesized a series of peptidyl fluoroketones that reversibly inhibit the serine proteases human leukocyte elastase (HLE) and alpha-lytic protease (alpha-LP). Ac-ambo-AlaCF3 (1) inhibits HLE and alpha-LP with Ki's of 2.4 and 15 mM, respectively. The effects of structural variations on this parent compound on Ki and the kinetics of inhibition were studied. The acetyl group was replaced by the tripeptide Z-L-Ala-L-Ala-L-Pro to yield the tetrapeptide trifluoroketone (TFK) Z-L-Ala-L-Ala-L-Pro-ambo-AlaCF3 (2). This extension reduced Ki 3500-fold for HLE and 3000-fold for alpha-LP. Removal of a fluorine atom from a TFK decreases Ki about 15- to 30-fold with both enzymes. Replacement of one fluorine atom of 2 by a residue (-CH2-CH2-COLeuOMe) (6) which can interact with the S'1 and S'2 subsites decreased Ki 30-fold for HLE and 150-fold for alpha-LP compared to Z-L-Ala-L-Ala-L-Pro-ambo-AlaCF2H (3). The Ki of 6 for HLE is approximately equal to that of trifluoroketone 2. For alpha-LP Ki of 6 is 10-fold lower than that for the trifluoroketone 2. Inhibitors with Ki values less than 10(-7) M exhibit slow binding kinetics. By analogy to cholinesterases and chymotrypsin, it is likely that these enzymes combine with the keto form of the inhibitor to form the enzyme-inhibitor complex. Therefore, kon and Ki were corrected for the ketone concentration. The corrected kon values for the slow binding inhibitors are in most cases less than diffusion controlled, ranging between 8.2 X 10(4) and 4.68 X 10(6) M-1 s-1. An exception is Z-L-Ala-L-Ala-L-Pro-ambo-ValCF3 (8) where kon = 9 X 10(7) M-1 s-1, which is nearly diffusion controlled.     

In vitro evaluation of the antielastase activity of polycyclic β-lactams

A series of bi- and tricyclic β-lactam compounds was synthesized and evaluated as inhibitors of cleavage of synthetic substrates in vitro by the serine proteases Human Leukocyte Elastase (HLE), Human Leukocyte Proteinase 3 (HLPR3) and Porcine Pancreatic Elastase (PPE). The obtained results have permitted us to describe a homobenzocarbacephem compound as HLE and HLPR3 inhibitor, to observe the positive effect that the styryl group exerts on the HLE inhibitory activity in polycyclic β-lactam compounds and to conclude that the hydroxyl function decreases the HLE inhibitory activity or rules it out completely.     

Synthesis and in-vitro evaluation of novel low molecular weight thiocarbamates as inhibitors of human leukocyte elastase

A series of novel low molecular weight thiocarbamate esters (1e-6e) were synthesized and evaluated as inhibitors of human leukocyte elastase (HLE). The thiocarbamate esters studied consist of a substituted primary or secondary aliphatic or aromatic amine and a 1-phenyl-1H-tetrazole-5-thiol (Table I). The HLE catalyzed hydrolysis of N-methoxysuccinyl- L-Ala-L-Ala-L-Pro-L-Val-p-nitroanilide substrate was utilized as the measure of inhibition. N-n-butyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (1e) exhibited the highest inhibitory activity (k(obs) /[I] = 2.1 x 10(5) M(-1). min(-1) ) and N-allyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (2e) (K(obs) /[I] = 6.1 x 10(4) M(-1). min(-1) ) exhibited the second highest inhibitory activity of all the thiocarbamates. The aromatic N-phenyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (4e) showed the lowest inhibitory activity (K(obs) /[I] = 1.9 x 10(2) M(-1). min(-1) ) among the N-monosubstituted derivatives, similar to that of N-ethyl-N-n-butyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (5e) (K(obs) /[I] = 1.8 x 10(2) M(-1).min(-1) ). The N-isopropyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (3e) (K(obs) /[I] = 3.3 x 10(3) M(-1).min(-1) ) was about 10 fold more active than (4e) and N, N-diisopropyl, 1-phenyl-1H-tetrazole- 5-thiocarbamate (6e) showed no inhibitory activity against HLE. In the present work less than 3% of HLE specific activity was regained after 24 hours incubation with each of the tested N-monosubstituted thiocarbamates (1e-4e). The time-dependent inhibition of HLE by the thiocarbamate compounds (1e-5e) seems to involve the interaction and possible chemical modification of one enzyme residue. Straight chain nonpolar aliphatic substituents on the nitrogen of the thiocarbamate functionality may be essential for high inhibitory activity. As the degree of substitution (branching) on the nitrogen of the thiocarbamate functionality increases the inhibitory activity of the compounds decreases. The time-dependent inhibition of HLE and the slow deacylation rates by the N-monosubstituted thiocarbamates are consistent with irreversible inhibition.     

2,4,5-Triphenylisothiazol-3(2H)-one 1,1-dioxides as inhibitors of human leukocyte elastase

A series of substituted 2,4,5-triphenylisothiazol-3(2H)-one 1,1-dioxides 9 was synthesized and investigated as inhibitors of human leukocyte elastase (HLE). All compounds were found to inhibit HLE in a time-dependent manner and most of them exhibited k_obs/[I] values > 300 M^{? 1}s^{? 1}. The most potent 3-oxosultam of this series was 9l (k_obs/[I] = 2440 M^{? 1}s^{? 1}). Kinetic investigations performed with 9g and different substrate concentrations did not allow to clearly distinguish between a competitive or noncompetitive mode of inhibition. A more complex interaction is supported by the failure of a linear dependency of k_obs values on the inhibitor concentration.     

Utilization of the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold in the design of potent inhibitors of serine proteases: SAR studies using carboxylates

A series of carboxylate derivatives based on the 1,2,5-thiadiazolidin-3-one 1,1 dioxide and isothiazolidin-3-one 1,1 dioxide scaffolds has been synthesized and the inhibitory profile of these compounds toward human leukocyte elastase (HLE), cathepsin G (Cat G) and proteinase 3 (PR 3) was then determined. Most of the compounds were found to be potent, time-dependent inhibitors of elastase, with some of the compounds exhibiting k(inact)/K1 values as high as 4,928,300 M(-1) s(-1). The inhibitory potency of carboxylate derivatives based on the 1,2,5-thiadiazolidin-3-one 1,1 dioxide platform was found to be influenced by both the pKa and the inherent structure of the leaving group. Proper selection of the primary specificity group (R(I)) was found to lead to selective inhibition of HLE over Cat G, however, those compounds that inhibited HLE also inhibited PR 3, albeit less efficiently. The predictable mode of binding of these compounds suggests that, among closely-related serine proteases, highly selective inhibitors of a particular serine protease can be fashioned by exploiting subtle differences in their S' subsites. This study has also demonstrated that the degradative action of elastase on elastin can be abrogated in the presence of inhibitor 17.     

Protective effects of appropriate Zn<Superscript>2+</Superscript> levels against UVB radiation-induced damage in human lens epithelial cells in vitro

As one of the crucial factors of cataract formation, ultraviolet B (UVB) can lead to apoptosis of human lens epithelial cells. Zinc, a cell-protective metal against various toxic compounds, plays an important role in protecting target cells from damage. Nevertheless, it is still unclear whether zinc exhibits protective effect on human lens epithelial cells (HLE B-3) against UVB-induced damage. In this study, we investigated the protective effect of zinc chloride (ZnCl₂) on UVB-induced HLE B-3 cell damage, explored the molecular mechanisms using real-time cell electronic sensing system, flow cytometry, real-time quantitative PCR and enzyme-linked immunosorbent assay techniques. The results show that ZnCl₂ is a potential inhibitor of UVB-induced HLE B-3 cell damage, and the underlying mechanisms are involved in decreasing the overproduction of reactive oxygen species and mitochondrial dysfunction, promoting intracellular calcium homeostasis recovery, and thus maintaining cell normal physiological functions. Taken together, our findings suggest that appropriate zinc levels have potential for protecting HLE B-3 cells against UVB-induced damage, and this finding may be clinically useful.     

Specificity, stability, and potency of monocyclic beta-lactam inhibitors of human leucocyte elastase.

Stable, potent, highly specific, time-dependent monocyclic beta-lactam inhibitors of human leucocyte elastase (HLE) are described. The heavily substituted beta-lactams are stable under physiological conditions including in the presence of enzymes of the digestive tract. The beta-lactams were unstable in base. At pH 11.3 and 37 degrees C they were hydrolyzed with half-lives of 1.5-2 h. Hydrolysis produced characteristic products including the substituent originally at C-4 of the lactam ring, a substituted urea, and products resulting from decarboxylation of the acid after ring opening. The most potent beta-lactam displayed only 2-fold less activity versus HLE than alpha 1PI, the natural proteinaceous inhibitor. The compounds were more potent against the human and primate PMN elastases than versus either the dog or rat enzymes. Differences in the structure-activity relationships of the human versus the rat enzymes suggest significant differences between these two functionally similar enzymes. The specificity of these compounds toward HLE versus porcine pancreatic elastase (PPE) is consistent with the differences in substrate specificity reported for these enzymes [Zimmerman & Ashe (1977) Biochim. Biophys. Acta 480, 241-245]. These differences suggest that the alkyl substitutions at C-3 of the lactam ring bind in the S1 specificity pocket of these enzymes. The dependence of the stereochemistry at C-4 suggests additional differences between HLE and PPE. Most of the compounds do not inhibit other esterases or human proteases. Weak, time-dependent inhibition of human cathepsin G and alpha-chymotrypsin by one compound suggested a binding mode to these enzymes that places the N-1 substitution in the S1 pocket.     

6-Acylamino-2-1[(ethylsulfonyl)oxy]-1H-isoindole-1,3-diones mechanism-based inhibitors of human leukocyte elastase and cathepsin G: effect of chirality in the 6-acylamino substituent on inhibitory potency and selectivity

Inhibition of human leukocyte elastase(HLE) by a series of 6-acylamino-2-[(ethylsulfonyl)oxy)]-1H-isoindole-1,3-diones was determined and compared to their inhibition of ChT, PPE, and Cat G. The best inhibitor of the series was 6-((1'S)-camphanyl)amino-2-[(ethylsulfonyl) oxy]-1H-isoindole-1,3-dione 5b, with a k(obs)/[I] = 11,000 M(-1) s(-1). This study revealed that HLE shows a preference for the S stereochemistry and tolerates hydrophobic substituents in the Sn' binding sites. Molecular modeling of non-covalent HLE-inhibitor complexes was used as a tool to investigate our binding model. Buffer stability assays reveal that these compounds are susceptible to hydrolysis at physiological pH.     

50th anniversary of the word "allosteric"

A brief historical account on the origin and meaning of the word "allosteric" is presented. The word was coined in an attempt to qualify the chemical mechanism of the feedback inhibition of bacterial enzymes by regulatory ligands. The data lead to the proposal that, at variance with the classical mechanism of mutual exclusion by steric hindrance, the inhibition takes place through an "allosteric" interaction between "no overlapping", stereospecifically distinct, sites for substrate and feedback inhibitor, mediated by a discrete reversible alteration of the molecular structure of the protein.     

The HLE hybrid dysgenesis syndrome in the midgeChironomus thummi: Differences in the dysgenic potential between strains

After crossing experiments between twoChironomus subspecies the HLE hybrid dysgenesis syndrome occurs non-reciprocally in the F1 hybrids if the males of each of the fourCh. th. thummi strains (HL, So, G, Kr) are crossed to females ofCh. th. piger, strain E. In hybrid egg masses, differences in the strength of the abnormal trait reduced egg hatch were found between the differentpiger E ×thummi interstrain crosses. From the results it is concluded that eachthummi strain has a specific hybrid dysgenesis potential which determines the severity of the HLE syndrome in the hybrids. The HLE syndrome occurs also in the hybrids of crosses of differentCh. th. thummi strains. In these hybrids the syndrome is only obtained if the male parent descends from a strain which has a higher HLE hybrid dysgenesis potential than that strain from which the female parent is derived. These results demonstrate that the HLE syndrome is not restricted to the hybrids of thethummi × piger subspecies cross. In theCh. th. thummi interstrain crosses the hybrids of that cross direction are normal, which is reciprocal to the cross direction affected by the HLE syndrome. This is in contrast to thethummi × piger subspecies crosses, where the hybrids of one cross direction always show the HLE syndrome and the hybrids of the reciprocal cross direction the Rud syndrome. Thus, the present study also demonstrates that the HLE and Rud hybrid dysgenesis syndrome inChironomus are independent from one another.