The role of Tyr605 and Ala607 of thimet oligopeptidase and Tyr606 and Gly608 of neurolysin in substrate hydrolysis and inhibitor binding

The physicochemical properties of TOP (thimet oligopeptidase) and NEL (neurolysin) and their hydrolytic activities towards the FRET (fluorescence resonance energy transfer) peptide series Abz-GFSXFRQ-EDDnp [where Abz is o-aminobenzoyl; X=Ala, Ile, Leu, Phe, Tyr, Trp, Ser, Gln, Glu, His, Arg or Pro; and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] were compared with those of site-mutated analogues. Mutations at Tyr605 and Ala607 in TOP and at Tyr606 and Gly608 in NEL did not affect the overall folding of the two peptidases, as indicated by their thermal stability, CD analysis and the pH-dependence of the intrinsic fluorescence of the protein. The kinetic parameters for the hydrolysis of substrates with systematic variations at position P1 showed that Tyr605 and Tyr606 of TOP and NEL respectively, played a role in subsite S1. Ala607 of TOP and Gly608 of NEL contributed to the flexibility of the loops formed by residues 600-612 (GHLAGGYDGQYYG; one-letter amino acid codes used) in NEL and 599-611 (GHLAGGYDAQYYG; one-letter amino acid codes used) in TOP contributing to the distinct substrate specificities, particularly with an isoleucine residue at P1. TOP Y605A was inhibited less efficiently by JA-2 {N-[1-(R,S)-carboxy-3-phenylpropyl]Ala-Aib-Tyr-p-aminobenzoate}, which suggested that the aromatic ring of Tyr605 was an important anchor for its interaction with wild-type TOP. The hydroxy groups of Tyr605 and Tyr606 did not contribute to the pH-activity profiles, since the pKs obtained in the assays of mutants TOP Y605F and NEL Y606F were similar to those of wild-type peptidases. However, the pH-kcat/Km dependence curve of TOP Y605A differed from that of wild-type TOP and from TOP Y606F. These results provide insights into the residues involved in the substrate specificities of TOP and NEL and how they select cytosolic peptides for hydrolysis.     

Mitochondrial DNA effects on fitness in Drosophila subobscura

We tested different fitness components on a series of conspecific mtDNA haplotypes, detected by RFLPs in Drosophila subobscura. Additionally, haplotype VIII, endemic to the Canary Islands, was tested upon its own native nuclear DNA background and upon that of the rest of mtDNAs tested herein. We found that both nuclear and mitochondrial DNA can have a significant effect upon their hosts' fitness, and that negative selection is one of the mechanisms that can intervene in this species' mtDNA haplotype pattern. We discuss the importance of this mechanism in relation to genetic drift, in the form of periodic population bottlenecks, and how the latter can enhance the former. We also detected a significant positive effect of haplotype VIII upon fitness that could explain in part the dominance of this endemic haplotype on some of the Canary Islands, and a mitochondrial heterosis involving this haplotype when on a foreign nuclear DNA background.     

In vitro activity of the hydroethanolic extract and biflavonoids isolated from Selaginella sellowii on Leishmania (Leishmania) amazonensis

This study is the first phytochemical investigation of Selaginella sellowii and demonstrates the antileishmanial activity of the hydroethanolic extract from this plant (SSHE), as well as of the biflavonoids amentoflavone and robustaflavone, isolated from this species. The effects of these substances were evaluated on intracellular amastigotes of Leishmania (Leishmania) amazonensis, an aetiological agent of American cutaneous leishmaniasis. SSHE was highly active against intracellular amastigotes [the half maximum inhibitory concentration (IC50) = 20.2 µg/mL]. Fractionation of the extract led to the isolation of the two bioflavonoids with the highest activity: amentoflavone, which was about 200 times more active (IC50 = 0.1 μg/mL) and less cytotoxic than SSHE (IC50 = 2.2 and 3 μg/mL, respectively on NIH/3T3 and J774.A1 cells), with a high selectivity index (SI) (22 and 30), robustaflavone, which was also active against L. amazonensis (IC50 = 2.8 µg/mL), but more cytotoxic, with IC50 = 25.5 µg/mL (SI = 9.1) on NIH/3T3 cells and IC50 = 3.1 µg/mL (SI = 1.1) on J774.A1 cells. The production of nitric oxide (NO) was lower in cells treated with amentoflavone (suggesting that NO does not contribute to the leishmanicidal mechanism in this case), while NO release was higher after treatment with robustaflavone. S. sellowii may be a potential source of biflavonoids that could provide promising compounds for the treatment of cutaneous leishmaniasis.     

Distribution of Recombination Hotspots in the Human Genome – A Comparison of Computer Simulations with Real Data

Recombination is the main cause of genetic diversity. Thus, errors in this process can lead to chromosomal abnormalities. Recombination events are confined to narrow chromosome regions called hotspots in which characteristic DNA motifs are found. Genomic analyses have shown that both recombination hotspots and DNA motifs are distributed unevenly along human chromosomes and are much more frequent in the subtelomeric regions of chromosomes than in their central parts. Clusters of motifs roughly follow the distribution of recombination hotspots whereas single motifs show a negative correlation with the hotspot distribution. To model the phenomena related to recombination, we carried out computer Monte Carlo simulations of genome evolution. Computer simulations generated uneven distribution of hotspots with their domination in the subtelomeric regions of chromosomes. They also revealed that purifying selection eliminating defective alleles is strong enough to cause such hotspot distribution. After sufficiently long time of simulations, the structure of chromosomes reached a dynamic equilibrium, in which number and global distribution of both hotspots and defective alleles remained statistically unchanged, while their precise positions were shifted. This resembles the dynamic structure of human and chimpanzee genomes, where hotspots change their exact locations but the global distributions of recombination events are very similar.     

The peptide repertoires of HLA-B27 subtypes differentially associated to spondyloarthropathy (B*2704 and B*2706) differ by specific changes at three anchor positions

HLA-B*2704 is strongly associated with ankylosing spondylitis. B*2706, which differs from B*2704 by two amino acid changes, is not associated with this disease. A systematic comparison of the B*2704- and B*2706-bound peptide repertoires was carried out to elucidate their overlap and differential features and to correlate them with disease susceptibility. Both subtypes shared about 90% of their peptide repertoires, consisting of peptides with Arg(2) and C-terminal aliphatic or Phe residues. B*2706 polymorphism influenced specificity at three anchor positions: it favored basic residues at P3 and POmega-2 and impaired binding of Tyr and Arg at POmega. Thus, the main structural feature of peptides differentially bound to B*2704 was the presence of C-terminal Tyr or Arg, together with a strong preference for aliphatic/aromatic P3 residues. This is the only known feature of B*2704 and B*2706 that correlates to their differential association with spondyloarthropathy. The concomitant presence of basic P3 and POmega-2 residues was observed only among peptides differentially bound to B*2706, suggesting that it impairs binding to B*2704. Similarity between peptide overlap and the degree of cross-reaction with alloreactive T lymphocytes suggested that the majority of shared ligands maintain unaltered antigenic features in the context of both subtypes.     

A simple cell labeling technique by means of lectins linked to fluorochromes for the detection of cells on tissue sections

Summary— We designed a protocol for cell labeling with the lectin wheat germ agglutinin (WGA) linked to the fluorochrome tetramethyl-rhodamine isothiocyanate (TRITC) for effective detection of the B16F10 melanoma and Lewis lung carcinoma (LLc) cells on pulmonary histological sections from C57BL6; mice. We have also determined a suitable concentration of WGA-TRITC (10 μg/ml), which leads to a very intense and homogeneous labeling of the cells, as it avoids cell clumping due to the presence of the lectin WGA. In order to determine to what extent the method affects these tumor cells, we have studied some important aspects related to their metastatic behavior, taking into account three parameters: a) viability and rate of proliferation of the cells cultured in vitro; b) percentage of animals C57BL6 mice) bearing metastasis 15 days after intravenous inoculation with 10⁵ B16F10 or LLc cells; and c) pattern of distribution of tumor foci in lung. There were no significant differences in these three parameters between the WGA-TRITC labeled-cells compared to the cultures of non-labeled cells in either of the cell lines (B16F10, LLc). Thus, we conclude that B16F10 and LLc tumor cells can be labeled following the protocol set-up in our study, as it allows these cells to be neatly identified on tissue sections and it causes no important physiological changes in the cells, with regard to metastatic behavior. These points make this technique very suitable for the detection of B16F10 and LLc cells on histological sections in studying their behavior during the first stages of the metastatic process.     

Different Characteristics of the Two Glutamate Synthases in the Green Leaves of Lycopersicon esculentum

The two glutamate synthases, NAD(P)H- and ferredoxin-dependent, from the green leaves of tomato plants (Lycopersicon esculentum L. cv Hellfrucht frühstamm) differed in their chemical properties and catalytic behavior. Gel filtration of NAD(P)H enzyme gave an apparent molecular size of 158 kilodalton, whereas the ferredoxin enzyme molecular size was 141 kilodalton. Arrhenius plots of the activities of the two enzymes showed that the NAD(P)H enzyme had two activation energies; 109.6 and 70.5 kilojoule per mole; the transition temperature was 22°C. The ferredoxin enzyme however, had only one activation energy; 56.1 kilojoule per mole. The respective catalytic activity pH optima for the NAD(P)H- dependent and the ferredoxin dependent enzymes were around 7.3 and 7.8. In experiments to evaluate the effects of modulators aspartate enhanced the NAD(P)H-linked activity, with a K_a value of 0.25 millimolar, but strongly inhibited that of the ferredoxin-dependent glutamate synthase with a K_i of 0.1 millimolar. 3-Phosphoserine was another inhibitor of the ferredoxin dependent enzyme with a K_i value of 4.9 millimolar. 3-Phosphoglyceric acid was a potent inhibitor of the ferredoxin-dependent form, but hardly affected the NAD(P)H-dependent enzyme. The results are discussed and interpreted to propose different specific functions that these activities may have within the leaf tissue cell.     

Production and characterization of human renin antibodies with region-oriented synthetic peptides

Polyclonal and monoclonal antibodies were raised against pure human renin, but nothing was known about the regions against which they were directed. Using a three-dimensional model of mouse submandibular renin, we selected seven peptide sequences as belonging to potential epitopes. The main criteria for their choice were the location of the peptide sequences near the catalytic region and on the surface of the renin molecule and their hydrophilicity. After transposition of the regions to the 340-amino acid sequence of human renin, the seven peptides (corresponding to amino acids 50-60, 63-71, 81-90, 118-126, 162-169, 247-255, and 287-295) were synthesized, coupled to bovine serum albumin, and injected into rabbits. Five of these peptides elicited antibodies, and 50-68% binding of the corresponding iodinated peptide was obtained with a 1:25 dilution of antiserum. The antisera titers ranged from 1:5,000 to 1:100,000 when tested by enzyme-linked immunosorbent assay. The same antisera bound 15-65% of labeled pure human renin at a final dilution of 1:2.5, the highest percentage being obtained with peptide 81-90 antiserum. At a 1:5 dilution, the five antisera inhibited renin activity by 23-68% in human plasma with a high renin activity (40 ng of angiotensin I/h/ml). At a final dilution of 1:50, peptide 81-90 antiserum was still capable of producing 25% inhibition. Purified IgG (0.6 mg) from this antiserum inhibited pure human renin activity by up to about 40%, as measured by its reaction with pure synthetic human tetradecapeptide substrate. Antigenic peptides that mimic a part of the human renin sequence, especially peptide 81-90 representing the "flap" covering the cleft between the two renin lobes, constitute promising tools for the development of a synthetic antirenin vaccine.     

Evolutionary Implications of Dental Eruption in Dasypus (Xenarthra)

Late eruption of the permanent dentition was recently proposed as a shared anatomical feature of endemic African mammals (Afrotheria), with anecdotal reports indicating that it is also present in dasypodids (armadillos). In order to clarify this question, and address the possiblity that late eruption is shared by afrotherians and dasypodids, we quantified the eruption of permanent teeth in Dasypus, focusing on growth series of D. hybridus and D. novemcinctus. This genus is the only known xenarthran that retains two functional generations of teeth. Its adult dentition typically consists of eight upper and eight lower ever-growing (or euhypsodont) molariforms, with no premaxillary teeth. All but the posterior-most tooth are replaced, consistent with the identification of a single molar locus in each series. Comparison of dental replacement and skull metrics reveals that most specimens reach adult size with none or few erupted permanent teeth. This pattern of growth occurring prior to the full eruption of the dentition is similar to that observed in most afrotherians. The condition observed in Dasypus and many afrotherians differs from that of most other mammals, in which the permanent dentition erupts during (not after) growth, and is complete at or near the attainment of sexual maturity and adult body size. The suture closure sequence of basicranial and postcranial epiphyses does not correlate well with dental eruption. The basal phylogenetic position of the taxon within dasypodids suggests that diphyodonty and late dental replacement represent the condition of early xenarthrans. Additionally, the inferred reduction in the number of molars to a single locus and the multiplication of premolars represent rare features for any living mammal, but may represent apomorphic characters for Dasypus.     

Donepezil-tacrine hybrid related derivatives as new dual binding site inhibitors of AChE

A new series of donepezil–tacrine hybrid related derivatives have been synthesised as dual acetylcholinesterase inhibitors that could bind simultaneously to the peripheral and catalytic sites of the enzyme. These new hybrids combined a tacrine, 6-chlorotacrine or acridine unit as catalytic binding site and indanone (the heterocycle present in donepezil) or phthalimide moiety as peripheral binding site of the enzyme, connected through a different linker tether length. One of the synthesised compounds emerged as a potent and selective AChE inhibitor, which is able to displace propidium in a competition assay. These results seem to confirm the ability of this inhibitor to bind simultaneously to both sites of the enzyme and make it a promising lead for developing disease-modifying drugs for the future treatment of Alzheimer’s disease. To gain insight into the molecular determinants that modulate the inhibitory activity of these compounds, a molecular modelling study was performed to explore their binding to the enzyme.