Tryptophan Biosynthesis and Production of Other Related Compounds from Indole and L-Serine by Mixed Ruminal Bacteria, Protozoa, and Their Mixture In Vitro

Tryptophan (Trp) biosynthesis and production of other related compounds from 1 mM each of indole (IND), L-serine (Ser), and IND plus Ser by mixed ruminal bacteria (B), protozoa (P), and their mixture (BP) in an in vitro system were quantitatively investigated. Ruminal microorganisms were anaerobically incubated at 39°C for 12 h. Trp and other related compounds produced in both the supernatants and microbial hydrolyzates of the incubation were analyzed by HPLC. B, P, and BP suspensions were not able to produce Trp when incubated with only IND or Ser. Appreciable amounts of Trp (9.8, 3.1, and 6.6% of substrate) were produced from IND plus Ser after 12 h by B, P, and BP suspensions, respectively. Trp produced from IND + Ser in B was found only in the hydrolyzate, whereas it was found in the medium as a free form in P after a 12-h incubation period. Rumen bacteria and protozoa were separately demonstrated for the first time to produce Trp from IND plus Ser, and the ability of P to produce Trp from IND plus Ser was about one-third that of B in 12 h. Trp produced from IND plus Ser by B, P, and BP suspensions was simultaneously degraded into its related compounds, and, among them, indoleacetic acid (IAA) was a major product found in B. Production of IAA was 4.3, 0.3, and 3.2% of IND in 12 h by B, P, and BP suspensions, respectively. A small amount of skatole (SKT) (1.1 and 2.5% in B and BP, respectively) and p-cresol (CRL) (2.4 and 3.4% in B and BP, respectively) were also produced from IND plus Ser during 12-h incubation. P suspension produced no SKT or CRL from IND plus Ser in 12-h incubation. These results suggested for the first time that both rumen bacteria and protozoa have an ability to produce Trp from IND plus Ser, and the ability was higher in B than in P. The ratios of Trp produced from IND plus Ser to that from indolepyruvic acid by B, P, and BP were 1:3.4, 1:14.2, and 1:6.6 during 12-h incubation period. From these results, the degree of importance of producing Trp from IND plus Ser in the rumen was indicated. Received: 18 February 1999 / Accepted: 18 May 1999     

Separation of tryptophan pyrrolooxygenase into three molecular forms. A study of their substrate specificities using tryptophyl-containing peptides and proteins

Tryptophan pyrrolooxygenase from wheat germ was separated into three molecular forms by microgranular DEAE-cellulose using a stepwise or a linear gradient elution procedure. In the first case molecular forms A and B were eluted with 10 mM Tris/HCl buffer (pH 7.4) and molecular form C was eluted with 50 mM KCl in the same buffer. The same separation could also be achieved with a linear KCl gradient (0-100 mM) in 10 mM Tris/HCl buffer (pH 7.4). The three molecular forms of tryptophan pyrrolooxygenase oxidized L-, D-, DL-Trp as well as many Trp derivatives with formation of N-formylkynurenyl derivatives. They also efficiently oxidized Trp-Phe, Trp-Tyr, Trp-Ala, Ala-Trp, Trp-Gly, Gly-Trp, Trp-Leu, Leu-Trp, Pro-Trp and Val-Trp, although the dipeptides were oxidized at different rates by the three molecular forms. A number of tryptophyl-containing tetra-, penta-, octa-, nona- and decapeptides were also oxidized. The oligopeptides which were known to have a helical conformation were better substrates than the smaller oligopeptides which were devoid of the conformational factor. The three molecular forms of tryptophan pyrrolooxygenase oxidized the tryptophyl residues of lysozyme, pepsin, chymotrypsin, trypsin and bovine serum albumin. It was found that molecular form A oxidized the more exposed (or hydrophilic) Trp residues of the proteins, while molecular form C also oxidized the Trp residues of a more hydrophobic nature. The three molecular forms were inhibited by chelating agents (alpha, alpha'-dipyridyl, EDTA and omicron-phenanthroline), although they differed in their sensitivities to these agents. Their optimum temperatures and inactivation rates at 65 degrees C was also different.     

Characterization and comparison of leuprolide degradation profiles in water and dimethyl sulfoxide.

The effect of solvent on the rate of leuprolide degradation and on the structure of the degradation products was explored. Leuprolide solutions (370 mg/mL) were prepared in water and dimethyl sulfoxide (DMSO) for delivery in DUROS osmotic implants. Both solvent systems demonstrated better than 90% stability after 1 year at 37 degrees C, where the DMSO formulation afforded better stability than the aqueous formulation and was used in subsequent clinical trials. The rate of leuprolide degradation in DMSO was also observed to accelerate with increasing moisture content, indicating that the aprotic solvent minimized chemical degradation. Interestingly, leuprolide degradation products varied with formulation vehicle. The proportions of leuprolide degradation products observed to form in water and DMSO at 37 degrees C were hydrolysis > aggregation > isomerization > oxidation and aggregation > oxidation > hydrolysis > isomerization, respectively. Specifically, more N-terminal hydrolysis and acetylation were observed under aqueous conditions, and increased Trp oxidation and Ser beta-elimination were seen under non-aqueous conditions. Furthermore, the major chemical degradation pathway changed with temperature in the DMSO formulation (decreasing oxidation with increasing temperature), but not in the aqueous formulation.     

Conformational and functional studies of gomesin analogues by CD, EPR and fluorescence spectroscopies

The aim of this work was to examine the bioactivity and the conformational behavior of some gomesin (Gm) analogues in different environments that mimic the biological membrane/water interface. Thus, manual peptide synthesis was performed by the solid-phase method, antimicrobial activity was evaluated by a liquid growth inhibition assay, and conformational studies were performed making use of several spectroscopic techniques: CD, fluorescence and EPR. [TOAC(1)]-Gm; [TOAC(1), Ser(2,6,11,15)]-Gm; [Trp(7)]-Gm; [Ser(2,6,11,15), Trp(7)]-Gm; [Trp(9)]-Gm; and [Ser(2,6,11,15), Trp(9)]-Gm were synthesized and tested. The results indicated that incorporation of TOAC or Trp caused no significant reduction of antimicrobial activity; the cyclic analogues presented a beta-hairpin conformation similar to that of Gm. All analogues interacted with negatively charged SDS both above and below the detergent's critical micellar concentration (cmc). In contrast, while Gm and [TOAC(1)]-Gm required higher LPC concentrations to bind to micelles of this zwitterionic detergent, the cyclic Trp derivatives and the linear derivatives did not seem to interact with this membrane-mimetic system. These data corroborate previous results that suggest that electrostatic interactions with the lipid bilayer of microorganisms play an important role in the mechanism of action of gomesin. Moreover, the results show that hydrophobic interactions also contribute to membrane binding of this antimicrobial peptide.     

Time-resolved fluorescence study of azurin variants: conformational heterogeneity and tryptophan mobility.

Time-resolved fluorescence and time resolved fluorescence anisotropy studies have been performed on wild-type azurin from Pseudomonas aeruginosa and two variants to study the mobility of Trp48. The two azurin variants in which the microenvironment of Trp48 was changed comprised the single mutations Ile7Ser and Phe110Ser. The experiments were performed on the holo-Cu(I), holo-Cu(II), and apo- forms at various pH values, viscosities, and temperatures; two distinct parts of the emission spectrum were selected for detection. Two prominent subnanosecond lifetimes in the fluorescence decays of the Cu(II) proteins could be observed. The decay of apo-azurin also consists of more than one component. The occurrence of more than one component in the fluorescence decays is explained by conformational heterogeneity. The anisotropy decay results appeared to be different for wild-type and mutated azurins. Phe110Ser and Ile7Ser azurin show more mobility of the Trp48 residue, as reflected in the order parameter.     

Characterization of pyrroloquinoline quinone amino acid derivatives by electrospray ionization mass spectrometry and detection in human milk

We describe a HPLC method coupled to electrospray ionization mass spectrometry (ESI/MS) for quantification and identification of pyrroloquinoline quinone (PQQ) and condensation products formed upon incubation of PQQ with amino acids (IPQ; imidazolopyrroloquinoline and I/OPQ/R; imidazolopyrroloquinoline with attached R-group). More importantly, using these methods we demonstrate the presence of both PQQ and IPQ in human milk in nanomolar to micromolar concentrations. PQQ was incubated with amino acids and condensation products were separated by HPLC. Fractions corresponding to each product were collected and molecular masses were determined using ESI/MS. Ala, Asp, Arg, Cys, Gly, Glu, Ser, Thr, Trp, and Tyr form IPQ upon incubation with PQQ. Yields of IPQ were low (<5%) for Asp and Glu, yet high (>60%) for Thr. In addition to IPQ, Ala, Arg, Cys, Ser, Trp, and Tyr formed IPQ/R derivatives. His, Ile, Leu, Glu, Leu, Lys, Met, and Phe form only IPQ/R derivatives. Proline did not react with PQQ. Mass spectra indicate that PQQ forms stable hydrated carbonyls and decarboxylates easily. Although mass spectra were complicated by the oxidation state of the quinone and decarboxylation of PQQ, these methods are invaluable for the rapid detection of the full range of PQQ adducts in biological matrices.     

Alteration of the iron-sulfur cluster composition of Escherichia coli dimethyl sulfoxide reductase by site-directed mutagenesis.

We have used site-directed mutagenesis to alter the [Fe-S] cluster composition of Escherichia coli dimethyl sulfoxide (DMSO) reductase (DmsABC). The electron-transfer subunit (DmsB) of this enzyme contains 16 Cys residues arranged in 4 groups (I-IV) which provide ligands to 4 [4Fe-4S] clusters [Cammack, R., & Weiner, J. H. (1990) Biochemistry 29, 8410-8416]. Strong homologies exist between these Cys groups and the four Cys groups of the electron-transfer subunit (NarH) of E. coli nitrate reductase (NarGHJI), which contains a [3Fe-4S] cluster in addition to multiple [4Fe-4S] clusters. The Cys group primarily involved in providing ligands to the [3Fe-4S] cluster of NarH has a Trp residue at a position equivalent to Cys102 of DmsB. We have mutated Cys102 to Trp, Ser, Tyr, and Phe and have investigated the altered enzymes in terms of their enzymatic activities and EPR properties. The mutant enzymes do not support electron transfer from menaquinol to DMSO, although they retain high rates of electron transport from reduced benzyl viologen to DMSO. The mutations cause major changes in the EPR properties of the enzyme in the fully reduced and oxidized states. In the oxidized state, new species are observed in all the mutants; these have spectral features comprising a peak at g = 2.03 (gz) and a peak-trough at g = 2.00 (gxy). The temperature dependencies, microwave power dependencies, and spin quantitations of these species are consistent with the Trp102, Ser102, Phe102, and Tyr102 mutations causing conversion of one of the [4Fe-4S] clusters present in the wild-type enzyme into [3Fe-4S] clusters in the mutant enzymes.     

The role of tryptophan residues in substrate binding to catalytic domains A and B of xylanase C from Fibrobacter succinogenes S85

Oxidation of the isolated catalytic domain B of xylanase C (XynC-B) from Fibrobacter succinogenes with N-bromosuccinimide (NBS) resulted in the modification of five of the seven Trp residues present in the enzyme. Hydrolytic activity of the enzyme was rapidly lost upon initiation of oxidation as a molar ratio of about two NBS molecules per molar equivalent of protein was sufficient to cause 50% inhibition of enzyme activity, and the addition of five molar equivalents of NBS resulted in less than 10% activity. Pre-incubation of XynC-B with the competitive inhibitor D-xylose resulted in the apparent protection of two Trp residues from oxidation. Xylose protection of the enzyme also resulted in a maintenance of activity, with 60% activity still evident after addition of 8-9 molar equivalents of NBS. This protection from inactivation was enhanced by the inclusion of xylohexaose in reaction mixtures. Under these conditions, however, a further Trp residue was protected from NBS oxidation. The three protected Trp residues were identified as Trp135, Trp161 and Trp202 by differential labelling and peptide mapping of NBS-oxidized preparations of the xylanase employing a combination of electrospray mass spectroscopic analysis and N-terminal sequencing. By analogy to the known structures of the family 11 xylanases, the fully conserved Trp202 residue is located on the only alpha-helix present in the enzymes, at the interface between it and the back of the beta-sheet which forms the active site cleft. Trp135 represents a highly conserved aromatic residue in family 11, but it is replaced with Thr in domain A of F. succinogenes xylanase C. To investigate the role of Trp135 in conferring the different activity profile of domain B relative to domain A, the Trp135Thr and Trp135Ala derivatives of domain B were prepared by site-directed mutagenesis. However, the kinetic parameters of the two domain B derivatives were not significantly different compared to the wild-type enzyme as reflected by K(M) and k(cat) values and product distribution profiles. Similar results were obtained with the Trp161Ala derivative of domain B, indicating that these two residues do not directly participate in the binding of substrate but likely form the foundation for binding subsite 2.     

Dimethyl sulfoxide induces both direct and indirect tau hyperphosphorylation

Dimethyl sulfoxide (DMSO) is widely used as a solvent or vehicle for biological studies, and for treatment of specific disorders, including traumatic brain injury and several forms of amyloidosis. As Alzheimer's disease (AD) brains are characterized by deposits of β-amyloid peptides, it has been suggested that DMSO could be used as a treatment for this devastating disease. AD brains are also characterized by aggregates of hyperphosphorylated tau protein, but the effect of DMSO on tau phosphorylation is unknown. We thus investigated the impact of DMSO on tau phosphorylation in vitro and in vivo. One hour following intraperitoneal administration of 1 or 2 ml/kg DMSO in mice, no change was observed in tau phosphorylation. However, at 4 ml/kg, tau was hyperphosphorylated at AT8 (Ser(202)/Thr(205)), PHF-1 (Ser(396)/Ser(404)) and AT180 (Thr(231)) epitopes. At this dose, we also noticed that the animals were hypothermic. When the mice were maintained normothermic, the effect of 4 ml/kg DMSO on tau hyperphosphorylation was prevented. On the other hand, in SH-SY5Y cells, 0.1% DMSO induced tau hyperphosphorylation at AT8 and AT180 phosphoepitopes in normothermic conditions. Globally, these findings demonstrate that DMSO can induce tau hyperphosphorylation indirectly via hypothermia in vivo, and directly in vitro. These data should caution researchers working with DMSO as it can induce artifactual results both in vivo and in vitro.     

Preparation of C-6 substituted chitin derivatives under homogeneous conditions

Tosylation of chitin under homogeneous conditions was achieved by the reaction of tosyl chloride with chitin in a DMAc/LiCl solvent system. The resultant tosyl-chitin was fully N-acetylated with acetic anhydride in methanol. The fully acetylated tosyl-chitin was subsequently reacted with the sodium salts of ethyl p-hydroxybenzoate, diethyl malonate, and diethyl phosphite in DMAc to give the corresponding chitin derivatives of 6-O-ethyl benzoate-chitin, 6-deoxy-diethyl malonate-chitin, and 6-(deoxydiethyl) phosphite-chitin, respectively. Subsequent hydrolysis of the chitin-ester derivatives with tert-butoxide in dimethyl sulfoxide (DMSO) generated 6-O-carboxyphenyl-chitin and 6-(deoxydicarboxy)methyl-chitin. The structures of the chitin derivatives were assessed by FT-IR, (13)C NMR, and (31)P NMR, while the degree of substitution of the S(N)2 reaction was estimated by elemental analysis. All the chitin derivatives were found to be soluble or swellable in water, DMAc, or DMSO.     

Tryptophan residue of Trp-Ser-X-Trp-Ser motif in extracellular domains of erythropoietin receptor is essential for signal transduction.

The Trp-Ser-X-Trp-Ser motif commonly exists just outside the transmembrane domains of all cytokine receptors so far isolated. The role of this conserved motif in erythropoietin receptor was examined by assessing a series of mutant receptors on erythropoietin-induced signal transduction. Replacement of one of the two conserved Trp residues in the motif to Gly was found to completely abolish the binding of erythropoietin to the receptor and also to lose the ability to transduce the factor-dependent growth signal. While the mutants with one Ser residue converted to Gly or Ala retained full biological activities, the replacement of both conserved Ser residues diminished the functions of the receptor. Furthermore, the receptors lacking a part or all of the Trp-Ser-X-Trp-Ser motif did not respond to erythropoietin. The Trp-Ser-X-Trp-Ser motif, especially Trp residue, located in extracellular domains of the erythropoietin receptor thus appears to play a critical role in receptor-mediated signal transduction.     

Suppression of the serT42 mutation with modified tRNA(1Ser) and tRNA(5Ser) genes.

Serine tRNA gene derivatives with altered anticodons were introduced to the temperature-sensitive serT42 mutant, whose tRNA(1Ser) shows a base substitution of A10 for wild type G10. When a low copy number vector-system was used, the growth and beta-galactosidase synthetic activity of the serT42 mutant were restored by complementation with the tRNA(5Ser) (T34) gene or the tRNA(1Ser) (G34) gene as well as the tRNA(1Ser) (wt) gene, but not with tRNA(5Ser) (wt), tRNA(1Ser) (A34) or tRNA(1Ser) (C34) genes at 42 degrees C. When multicopy vectors were used, the transformation even with tRNA(1Ser) (A10) gene restored the growth and beta-galactosidase synthetic activity at 42 degrees C. The tRNA(1Ser) (A10) showed no thermosensitivity in serine acceptor activity by in vitro assay. At 42 degrees C, the amount of tRNA(1Ser) (A10) in the serT42 mutant was almost the same as those in the wild type. The nucleotides in the tRNA(1Ser) (A10) were found to be fully modified like those in the wild type tRNA(1Ser). Both of the tRNAs transcribed from tRNA(5Ser) (T34) and tRNA(1Ser) (G34) genes showed serine acceptor activity. Modified nucleosides of these tRNAs were also analyzed.     

A second mutation associated with apparent beta-hexosaminidase A pseudodeficiency: identification and frequency estimation.

Deficient activity of beta-hexosaminidase A (Hex A), resulting from mutations in the HEXA gene, typically causes Tay-Sachs disease. However, healthy individuals lacking Hex A activity against synthetic substrates (i.e., individuals who are pseudodeficient) have been described. Recently, an apparently benign C739-to-T (Arg247Trp) mutation was found among individuals with Hex A levels indistinguishable from those of carriers of Tay-Sachs disease. This allele, when in compound heterozygosity with a second "disease-causing" allele, results in Hex A pseudodeficiency. We examined the HEXA gene of a healthy 42-year-old who was Hex A deficient but did not have the C739-to-T mutation. The HEXA exons were PCR amplified, and the products were analyzed for mutations by using restriction-enzyme digestion or single-strand gel electrophoresis. A G805-to-A (Gly269Ser) mutation associated with adult-onset GM2 gangliosidosis was found on one chromosome. A new mutation, C745-to-T (Arg249Trp), was identified on the second chromosome. This mutation was detected in an additional 4/63 (6%) non-Jewish and 0/218 Ashkenazi Jewish enzyme-defined carriers. Although the Arg249Trp change may result in a late-onset form of GM2 gangliosidosis, any phenotype must be very mild. This new mutation and the benign C739-to-T mutation together account for approximately 38% of non-Jewish enzyme-defined carriers. Because carriers of the C739-to-T and C745-to-T mutations cannot be differentiated from carriers of disease-causing alleles by using the classical biochemical screening approaches, DNA-based analyses for these mutations should be offered for non-Jewish enzyme-defined heterozygotes, before definitive counseling is provided.     

Active site heterogeneity in dimethyl sulfoxide reductase from Rhodobacter capsulatus revealed by Raman spectroscopy

Raman spectroscopy has been used to investigate the structure of the molybdenum cofactor in DMSO reductase from Rhodobacter capsulatus. Three oxidized forms of the enzyme, designated 'redox cycled', 'as prepared', and DMSOR(mod)D, have been studied using 752 nm laser excitation. In addition, two reduced forms of DMSO reductase, prepared either anaerobically using DMS or using dithionite, have been characterized. The 'redox cycled' form has a single band in the Mo=O stretching region at 865 cm(-1) consistent with other studies. This oxo ligand is found to be exchangeable directly with DMS(18)O or by redox cycling. Furthermore, deuteration experiments demonstrate that the oxo ligand in the oxidized enzyme has some hydroxo character, which is ascribed to a hydrogen bonding interaction with Trp 116. There is also evidence from the labeling studies for a modified dithiolene sulfur atom, which could be present as a sulfoxide. In addition to the 865 cm(-1) band, an extra band at 818 cm(-1) is observed in the Mo=O stretching region of the 'as prepared' enzyme which is not present in the 'redox cycled' enzyme. Based on the spectra of unlabeled and labeled DMS reduced enzyme, the band at 818 cm(-1) is assigned to the S=O stretch of a coordinated DMSO molecule. The DMSOR(mod)D form, identified by its characteristic Raman spectrum, is also present in the 'as prepared' enzyme preparation but not after redox cycling. The complex mixture of forms identified in the 'as prepared' enzyme reveals a substantial degree of active site heterogeneity in DMSO reductase.     

Dynamics of the protein structure of T169S pyranose 2‐oxidase in solution: Molecular dynamics simulation

Pyranose 2‐oxidase (P2O) from Trametes multicolor contains FAD as cofactor, and forms a tetramer. The protein structure of a mutated P2O, T169S (Thr169 is replaced by Ser), in solution was studied by means of molecular dynamics simulation and analyses of photoinduced electron transfer (ET) from Trp168 to excited isoalloxazine (Iso*), and was compared with wild type (WT) P2O. Hydrogen bonding between Iso and nearby amino acids was very similar as between T169S and WT protein. Distances between Iso and Tyr456 were extremely heterogeneous among the subunits, 1.7 (1.5 in WT) in subunit A (Sub A), 0.97 (2.2 in WT) in Sub B, 1.3 (2.1 in WT) in Sub C, 1.3 nm (2.0 in WT) in Sub D. Mean values of root of mean square fluctuation over all residues were greater by four times than those in WT. This suggests that the protein structure of T169S is much more flexible than that of WT. Electrostatic (ES) energies between Iso anion in one subunit and ionic groups in the entire protein were evaluated. It was found that more than 50% of the total ES energy in each subunit is contributed from other subunits. Reported fluorescence decays were analyzed by a method as WT, previously reported. Electron affinities of Iso* in T169S were appreciably higher than those in WT. Static dielectric constants near Iso and Trp168 were also quite higher in T169S than those in WT.     

Identification and functions of amino acid residues in PotB and PotC involved in spermidine uptake activity

Amino acid residues on PotB and PotC involved in spermidine uptake were identified by random and site-directed mutagenesis. It was found that Trp(8), Tyr(43), Trp(100), Leu(110), and Tyr(261) in PotB and Trp(46), Asp(108), Glu(169), Ser(196), Asp(198), and Asp(199) in PotC were strongly involved in spermidine uptake and that Tyr(160), Glu(172), and Leu(274) in PotB and Tyr(19), Tyr(88), Tyr(148), Glu(160), Leu(195), and Tyr(211) in PotC were moderately involved in spermidine uptake. Among 11 amino acid residues that were strongly involved in spermidine uptake, Trp(8) in PotB was important for insertion of PotB and PotC into membranes. Tyr(43), Trp(100), and Leu(110) in PotB and Trp(46), Asp(108), Ser(196), and Asp(198) in PotC were found to be involved in the interaction with PotD. Leu(110) and Tyr(261) in PotB and Asp(108), Asp(198), and Asp(199) in PotC were involved in the recognition of spermidine, and Trp(100) and Tyr(261) in PotB and Asp(108), Glu(169), and Asp(198) in PotC were involved in ATPase activity of PotA. Accordingly, Trp(100) in PotB was involved in both PotD recognition and ATPase activity, Leu(110) in PotB was involved in both PotD and spermidine recognition, and Tyr(261) in PotB was involved in both spermidine recognition and ATPase activity. Asp(108) and Asp(198) in PotC were involved in PotD and spermidine recognition as well as ATPase activity. These results suggest that spermidine passage from PotD to the cytoplasm is coupled to the ATPase activity of PotA through a structural change of PotA by its ATPase activity.     

Differential effects of FGFR2 mutations on syndactyly and cleft palate in Apert syndrome.

Apert syndrome is a distinctive human malformation characterized by craniosynostosis and severe syndactyly of the hands and feet. It is caused by specific missense substitutions involving adjacent amino acids (Ser252Trp or Pro253Arg) in the linker between the second and third extracellular immunoglobulin domains of fibroblast growth factor receptor 2 (FGFR2). We have developed a simple PCR assay for these mutations in genomic DNA, based on the creation of novel (SfiI) and (BstUI) restriction sites. Analysis of DNA from 70 unrelated patients with Apert syndrome showed that 45 had the Ser252Trp mutation and 25 had the Pro253Arg mutation. Phenotypic differences between these two groups of patients were investigated. Significant differences were found for severity of syndactyly and presence of cleft palate. The syndactyly was more severe with the Pro253Arg mutation, for both the hands and the feet. In contrast, cleft palate was significantly more common in the Ser252Trp patients. No convincing differences were found in the prevalence of other malformations associated with Apert syndrome. We conclude that, although the phenotype attributable to the two mutations is very similar, there are subtle differences. The opposite trends for severity of syndactyly and cleft palate in relation to the two mutations may relate to the varying patterns of temporal and tissue-specific expression of different fibroblast growth factors, the ligands for FGFR2.     

Polymorphism in the human 2'-5'-oligoadenylate synthetase genes (OAS), associated with predisposition to severe forms of tick-borne encephalitis, in populations from North Eurasia

2'-5'-oligoadenylate synthetases are a family of interferon-induced enzymes which play an important role in the antiviral defense in mammals. In human genome three genes encoding functional synthetases (OAS1, OAS2 and OAS3) form a cluster. Previously we found that particular genotypes and/or alleles of five single nucleotide polymorphisms (SNPs) located within OAS2 and OAS3 genes are associated with predisposition to severe forms of tick-borne encephalitis (TBE) in Russian population. In current study we investigated the distribution of three of that SNPs (OAS3rs2285932 (C/T Ile438Ile), OAS3rs2072136 (G/A, Ser567Ser) and OAS2 rs15895 (G/A, Trp720Ter relative to p71 isoform)) in seven populations from North Eurasia: Caucasians (Russians and Germans (from Altai region)), Central Asian Mongoloids (Altaians, Khakasses, Tuvinians and Shorians) and Arctic Mongoloids (Chukchi). Differences between populations in genotype, allele and haplotype frequencies and in linkage disequilibrium structure for these SNPs were detected. We found that these frequencies correlate with the ethnicity of the populations and with their supposed differential exposure to TBE virus. Particularly, the lowest frequencies of G/G genotype for OAS3 gene rs2072136 SNP (that according to our previously obtained data is associated with predisposition to severe forms of TBE) were found in Altaians, Khakasses, Tuvinians and Shorians who may highly contact with TBE virus in places of their habitation. Thus, data obtained allow to suppose that TBE virus might act as a selection factor for particular OAS genes variants in Central Asian Mongoloids.     

Structure-activity relationships of endothelin: importance of the C-terminal moiety

The vasoconstrictor activities of various forms of derivatives of endothelin (ET) were characterized in vitro by measuring the contraction of porcine coronary artery strips. The removal of the C-terminal Trp21 reduced the molar potency of the peptide by nearly 3 orders of magnitude. The removal of amino acid residues from the C-terminus of ET(1-20) further attenuated the activity. Replacement of Trp21 with D-Trp, reduction and carboxamidomethylation of the four Cys residues, or cleavage at Lys9 by lysyl endopeptidase all lowered the potency approximately 200 fold. While both native ET and [D-Trp21]ET induced a very slow and sustained vasoconstriction, the other derivatives of ET listed above showed a much more rapid kinetics of vasoconstriction. These results indicate that the C-terminal Trp of ET is especially important for the potent and extremely long-lasting vasoconstrictor activity characteristic to ET.