Bioinformatics in the post-genomic era

The Bioinformatics and Genome Research conference, one of the Cambridge Healthtech Institute's ‘Beyond the Genome’ conferences, was held 17–19 June 2001 in San Francisco, California, USA.     

Medicago truncatula, going where no plant has gone before

4th Workshop on Medicago truncatula, 7–10 July 2001, Madison WI, USA.     

Contents – Volume 53 (2001)

Volume Contents

Contents – Volume 53 (2001)     

Author index Volume 54 2001

Authors Index

Author index – Volume 53 (2001)     

Growth and development of Massaliatrema misgurni (Trematoda: Heterophyidae) in mice and its metacercarial morphology

The morphology of metacercariae of Massaliatrema misgurni Ohyama et al. (Ohyama et al., Parasitol Int 2001; 50; 267–71) was described, and their infectivity, egg output, growth and development in mice until day 35 post infection (PI) were studied. Metacercarial cysts from loaches imported from China to Japan were 199–349 μm in diameter and consisted of a very thick translucent outer layer and a refractile inner layer. Excysted metacercariae basically had the shape of miniature adults, and a pair of pre-developed testes but no other genital organs were recognized. The worm recovery rate from mice was 36.7–51.7% during days 3–7 PI, and decreased remarkably to 2.5 and 1.7% at days 28 and 35 PI. The prepatent period was 3–4 days, and the egg output quickly increased and sustained high levels at days 5–7 PI, then decreased suddenly at day 8 PI, and continued at a low level until day 28 PI. The size of the body and inner organs such as the oral sucker, pharynx, acetabulum, testes, ovary and seminal receptacle quickly increased until day 3 PI, and sustained at a plateau level until day 21 PI except testes which gradually decreased until 21 PI. The number of the uterine eggs increased with a short time lag compared to other genital organs and sustained a plateau level until day 21 PI. Compared with other Heterophyidae species, M. misgurni was characterized by the remarkably fast growth and development.     

Contribution of several nitrogen sources to growth of Alexandrium catenella during blooms in Thau lagoon, southern France

A monitoring program with a weekly sampling frequency over a 15-month period indicates that urea concentrations above a certain threshold level may trigger the blooms of Alexandrium catenella in Thau lagoon. However, urea concentrations were also sometimes related to ammonium and dissolved organic nitrogen concentrations, indicating that the role of urea may not be a direct one. An original approach is used to assess the relative contribution of several nitrogen sources (nitrate, nitrite, ammonium, urea) to growth of A. catenella by comparing nitrogen uptake rates to nitrogen-based growth rates estimated from dilution experiments during four blooms over a 4-year period (2001–2004) in Thau lagoon. Nitrate and nitrite contributed 0.1–14% and 0.1–5% respectively of growth requirements. Ammonium and urea were the main N sources fueling growth of A. catenella (30–100% and 2–59%, respectively). Indirect estimates indicated that an unidentified N source could also contribute significantly to growth at specific times. Concerning ammonium and urea uptake kinetics, half-saturation constants varied between 0.2 and 20 μgat N L⁻¹ for ammonium and between 0.1 and 44 μgat N L⁻¹ over the 4-year period, indicating that A. catenella can have a competitive advantage over other members of the phytoplankton even under low concentrations of ammonium and urea. However, the observed large changes in ammonium and urea uptake kinetics on a short time scale (days) during blooms preclude more precise estimates of those contributions to growth and require further investigation.     

Behaviour of Merinos divergently selected for multiple rearing ability in response to external stimuli

Data of Merino sheep descended from two divergent selection lines for (H line) and against (L line) multiple rearing ability were collected in three different trials to compare response to stressful situations between lines. Trial 1: the behaviour of 138, 20-month-old sheep born in 2001 and 124, 8-month-old sheep born in 2002 were observed in an arena test. Experimental animals amounted to 111 and 101 for the H line in the respective years. Corresponding numbers for the L line were 27 and 23, respectively. Progeny born in 2001 were accustomed to handling, as the tests were performed shortly after routine performance recording. Lambs born in 2002 were unaccustomed to handling when tested. Progeny from the H line born in 2002 allowed a smaller (P < 0.01) minimum distance between them and the human operator seated between them and a group of similar aged sheep than L line contemporaries. Progeny born in the L line during 2002 defecated more (P < 0.05) frequently than H line contemporaries. No line differences were found in progeny born during 2001. Trials 2 and 3 were designed to test whether the putative line difference in stress response would impact on physical meat quality. Trial 2: a total of 103, 18–20-month-old progeny from the two selection lines were slaughtered during 2001, 2002–2003. Mean slaughter weight, dressing percentage and carcass weight of H line animals were between 5 and 13% higher (P < 0.05) than that of L line animals. There were no differences in cooking loss, drip loss and tenderness between the two lines. As pertaining to the meat colour, L line progeny had less vivid red meat, as seen by their lower (P < 0.05) a* colour reading and chroma values. The L line animals had lower (P < 0.01) initial pH values as measured in the Musculus longissimus dorsi. These results are indicative of susceptibility to acute, short-term stress. Trial 3: 20 progeny of the two selection lines were slaughtered and their muscle pH measured at 11 intervals from 45 min to 48 h post slaughter. As in Trial 1, the initial pH of the L line animals were lower (P < 0.05) than that of H line animals. An opposite tendency was observed by 48 h post slaughter. The obtained results support a hypothesis that divergent selection for ewe multiple ability resulted in adaptations to cope with unfamiliar conditions.     

Sexual reproduction of the high mountain plant Saxifraga moschata Wulfen at varying lengths of the growing season

Flowering phenology, dynamics of seed development and reproductive success of Saxifraga moschata Wulfen, occurring from the alpine to the nival zone, was investigated in three climatically varying years on early- and late-thawing sites at 2340 m a.s.l. in the Austrian Alps. The growing season was particularly short in 2001, about normal in 2002 and exceptionally long and warm in 2003. S. moschata is a mid-season flowering species, which started to bloom about 1 month after snowmelt. From onset of anthesis until seed maturity an individual flower required 6–7 weeks, and all individuals of a site 9–11 weeks. In 2001, late flowering individuals lost about 40% of the seed crop because of too early snowfalls in autumn. No clear relationship could be found between thermal time and the length of different reproductive phases, which indicates that temperature was not a limiting factor for reproductive development in the growing seasons 2001–2003.

Plants from the earliest-thawing site produced about double the reproductive structures (reproductive shoots per individual, number of flowers per inflorescence, number of ovules per ovary), and had a significantly higher reproductive output (number of fruits and mature seeds per inflorescence) compared with individuals from the late-thawing site. Reproductive success was additionally affected by the seasonal climate and was lowest in the warm and dry summer of 2003. Fruit/flower ratio ranged between 0.6 and 0.8, seed/ovule ratio between 0.4 and 0.5 and relative reproductive success (RRS) between 0.3 and 0.4, depending on the site and year.

Different from our expectations, it took S. moschata relatively long to undergo reproductive development; however, its phenological plasticity and ability to continue seed maturation even under snow are most advantageous for a life at higher altitudes.     

A new member of YER057c family in Trypanosoma cruzi is adjacent to an ABC-transporter

Tcp17 is a Trypanosoma cruzi gene located contiguous to the ABC-transporter tcpgp2. The protein contains 160 amino acid residues with a predicted molecular mass of 16.5 kDa. Western blot analysis using a polyclonal antiserum against recombinant TCP17 revealed that the protein is only expressed in the epimastigote form of the parasite; we did not detect the protein either in the amastigote or trypomastigote forms. A sequence comparison of TCP17 showed a remarkable homology with a conserved family of prokaryotic and eukaryotic proteins called YER057c whose function has not yet been characterized. Here, we propose a new signature of this family considering the N-terminal: [IV]–X(4)–[AV]–[AP]–X–[AP]–X(3)–Y–X(9)–[LIVF]–X(2)–[SA]–G–[QS], and the C-terminal: [AT]–R–X(2)–[IVFY]–X–[VC]–X(2)–L–P–X(4)–[LIVM]–E–[IVM]–[DE] motifs. Immunofluorescence and immunoelectron microscopy studies suggest that the protein has a wide distribution in the cell, with a higher concentration in the external side of the plasma membrane, on the Golgi complex and on cytoplasmic vacuoles. Although the physiological function of TCP17 is unknown, its conservation in evolution suggests biological relevance in the parasite.     

Glucosamine modulates IL-1-induced activation of rat chondrocytes at a receptor level, and by inhibiting the NF-κB pathway

We recently reported that glucosamine reversed the decrease in proteoglycan synthesis and in UDP-glucuronosyltransferase I mRNA expression induced by interleukin-1β (IL-1β) [Arthritis Rheum. 44 (2001) 351–360]. In the present work, we show that glucosamine does not exert the same effects when chondrocytes were stimulated with reactive oxygen species (ROS). In order to better understand its mechanism of action, we determined if glucosamine could prevent the binding of IL-1β to its cellular receptors or could interfere with its signaling pathway at a post-receptor level. Addition of glucosamine to rat chondrocytes treated with IL-1β or with ROS decreased the activation of the nuclear factor κB, but not the activator protein-1. After treatment with IL-1β, glucosamine increased the expression of mRNA encoding the type II IL-1β receptor. These results emphasize the potential role of two regulating proteins of the IL-1β signaling pathway that could account for the beneficial effect of glucosamine in osteoarthritis.     

-Bungarotoxin binding to human muscle acetylcholine receptor: measurement of affinity, delineation of AChR subunit residues crucial to binding, and protection of AChR function by synthetic peptides

–Bungarotoxin (–BuTx) binds with high a.nity to the nicotinic acetylcholine receptor (AChR) of most species, mainly to sequences around the two cysteines at positions 192 and 193 of the –subunit, but other sequences of the –subunit and of the adjacent γ– or ε– and δ–subunits are also important in the native molecule. –BuTx binds strongly to human AChR but the short neurotoxins, for instance Erabutoxin B, are relatively ineffective at the human neuromuscular junction. In this article we compare the a.nity of ¹²⁵I––BuTx for Torpedo and human muscle AChR and the ability of neurotoxins to inhibit this binding. We examine the contribution to –BuTx binding of the three amino acids that differ between human and Torpedo AChR –185—199. In addition, we show that an –185—199, peptide that binds strongly to ¹²⁵I––BuTx and can inhibit its binding in solution, is also capable of protecting the AChR on a cell line or at the neuromuscular junction. Such peptides might be useful in the treatment of acute envenoming or the autoantibody–mediated block of AChR function that can occur in human disorders. © 1998 Elsevier Science Ltd. All rights reserved.     

Matching unrelated stimuli with same discriminative functions: training order effects

Previous research has shown that after training simple discriminations (A1+/A2−, B1+/B2−), bringing these tasks under conditional control (J1–A1, J2–A2) leads to transfer of discriminative control (J1+/J2−) and to generalized matching on the basis of same discriminative functions (e.g. J1–B1, J2–B2). The same occurs when conditional discriminations are trained (D1–E1, D2–E2; F1–G1, F2–G2). When the subjects are then trained to demonstrate correct relations (D1–E1, D2–E2) when given X1 and to demonstrate incorrect relations when given X2 (XD–E), transfer of discriminative control (X1+/X2−) and generalized matching on the basis of same discriminative functions emerges (e.g. X1F1–G1, X2F1–G2). The present study investigated if these performances are dependent on the training and/or testing order. In Experiment 1, the lower-order contingency tasks were trained before the higher-order contingency tasks (A1+/A2−, B1+/B2− before J–A, and D–E, F–G before XD–E). Half the subjects received the J–B test before the more complex XF–G test (Condition A), while for the other subjects, this testing order was reversed (Condition B). Finally, all subjects received additional tests in which they were given the opportunity to demonstrate the discriminative properties of the J and X stimuli (J1+/J2−, X1+/X2−), and to match the A, J, and X stimuli with newly introduced stimuli of same discriminative properties (e.g. J1-POLITE, J2-RUDE). Experiment 2 was the same except that the training order was reversed (J–A before A1+/A2−, B1+/B2−, and XD–E before D–E, F–G). The results were affected by the training order but not by the testing order. Transfer of discriminative functions and generalized matching on the basis of same functions only occurred reliably when the lower-order contingency tasks were trained first. A stimulus-control account of the data is offered.     

Simultaneous determination of felbamate and three metabolites in rat and dog plasmas by high-performance liquid chromatography

An isocratic liquid chromatographic method employing one extraction step and a 150 mm × 4.6 mm I.D. Spherisorb ODS2, 3-μm HPLC column using UV-absorbance detection at 210 nm has been developed for the quantitation of felbamate and three felbamate metabolites in 0.100-ml aliquots of rat and dog plasmas. The linear quantitation range in rat plasma is 0.195–200 μg/ml for felbamate; 1.563–200 μg/ml for the p-hydroxy metabolite; 0.391–200 μg/ml for the 2-hydroxy metabolite; and 0.098–200 μg/ml for the monocarbamate metabolite. The linear quantitation range in dog plasma is 0.195–200 μg/ml for felbamate; 0.781–200 μg/ml for the p-hydroxy metabolite; 0.195–200 μg/ml for the 2-hydroxy metabolite; and 0.098–200 μg/ml for the monocarbamate metabolite.     

Identification of amidated forms of GLP-1 in rat tissues using a highly sensitive radioimmunoassay

The development of a sensitive radioimmunoassay (RIA) for C-terminally amidated forms of glucagon-like peptide-1 (GLP-1) is described. Rabbits immunized with GLP-1(7–36)amide conjugated to bovine serum albumin with glutaraldehyde produced antisera containing high-affinity antibodies directed against an epitope that included the free amidated C-terminus of the peptide. These antisera could be used in a sensitive RIA (detection limit 0.1 fmol/tube) that measured GLP-1(7–36)amide and GLP-1(1–36)amide equally. Total concentrations of amidated GLP-1 immunoreactivity in extracts of rat hypothalamus, pancreas and intestine were determined by RIA, and resolved into GLP-1(7–36)amide, GLP-1(1–36)amide and unidentified cross-reacting substances by HPLC. Whereas only GLP-1(7–36)amide could be identified in the hypothalamus, in amounts that represented 55–94% of total glucagon-like immunoreactivity (GLI), the pancreas produced chiefly GLP-1(1–36)amide, representing 0.8–3.4% of total GLI, and only trace or undetectable amounts of GLP-1(7–36)amide (0–0.36% of total GLI). This argues against any role of intrapancreatic GLP-1(7–36)amide in the secretion of insulin. In the terminal ileum total amidated GLP-1 immunoreactivity represented 27–73% of total GLI, and in five of six specimens only GLP-1(7–36)amide could be identified on HPLC, in amounts representing 13–17% of total GLI. Only one specimen of terminal ileum contained HPLC-identified GLP-1(1–36)amide (13% of total GLI) in addition to GLP-1(7–36)amide (31% of total GLI). Acid–ethanol extraction of peptide-free rat plasma with added GLP-1(7–36)amide gave recoveries of 91±SEM 2% in the range 20–200 pmol/l. Basal plasma amidated GLP-1 in six unanaesthetized rats was 4.1±1.1 pmol/l and rose to a maximum of 15.4±3.0 pmol/l 10 min after intragastric glucose 1 g/kg, illustrating the modest level of plasma responses of amidated forms of GLP-1.     

[H]WAY–100635 for 5–HT1A receptor autoradiography in human brain: a comparison with [H]8–OH–DPAT and demonstration of increased binding in the frontal cortex in schizophrenia

WAY–100635 is the first selective, silent 5–HT_1A (5-hydroxytryptamine_1A, serotonin-1A) receptor antagonist. We have investigated the use of [³H]WAY–100635 as a quantitative autoradiographic ligand in post-mortem human hippocampus, raphe and four cortical regions, and compared it with the 5–HT_1A receptor agonist, [³H]8–OH–DPAT. Saturation studies showed an average Kd for [³H]WAY–100635 binding in hippocampus of 1.1 nM. The regional and laminar distributions of [³H]WAY–100635 binding and [³H]8–OH–DPAT binding were similar. The density of [³H]WAY–100635 binding sites was 60–70% more than that of [³H]8–OH–DPAT in all areas examined except the cingulate gyrus where it was 165% higher. [³H]WAY–100635 binding was robust and was not affected by the post-mortem interval, freezer storage time or brain pH (agonal state). Using [³H]WAY–100635, we confirmed an increase of 5–HT_1A receptor binding sites in the frontal cortex in schizophrenia, previously demonstrated with [³H]8–OH–DPAT. Compared to [³H]8–OH–DPAT, [³H]WAY–100635 has two advantages: it has a higher selectivity and affinity for the 5–HT_1A receptor, and it recognizes 5–HT_1A receptors whether or not they are coupled to a G-protein, whereas [³H]8–OH–DPAT primarily detects coupled receptors. Given these considerations, the [³H]WAY–100635 binding data in schizophrenia clarify two points. First, they indicate that the elevated [³H]8–OH–DPAT binding seen in the same cases is attributable to an increase of 5–HT_1A receptors rather than any other binding site. Second, the enhanced [³H]8–OH–DPAT binding in schizophrenia reflects an increased density of 5–HT_1A receptors, not an increased percentage of 5–HT_1A receptors which are G-protein-coupled. We conclude that [³H]WAY–100635 is a valuable autoradiographic ligand for the qualitative and quantitative study of 5–HT_1A receptors in the human brain.     

Effects of the gonadotropin-releasing hormone associated peptides (GAP) on the release of luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL) in vivo

Six peptide sequences residing between basic amino acid residues in GAP were tested for effects on the release of FSH, LH and PRL in vivo in ovariectomized, estrogen-progesterone-primed (OEP) rats. Synthetic GAP peptides (1–13, 1–23, 15–23, 25–36, 38–53 and 41–53) were injected intravenously (IV) into conscious OEP rats and plasma levels of FSH, LH and PRL were measured by RIA. The activity of GAP peptides in the control of PRL was further examined in ether-stressed male rats which were injected IV with GAP peptides just prior to a 1-min etherization. GAP(1–13) significantly stimulated FSH release at doses of 1, 10 and 100 μg, whereas it stimulated LH release only at the highest dose of 100 μg. GAP(1–23) elevated plasma levels of FSH and LH only at a dose of 100 μg. The other 4 peptides had no effect on the release of gonadotropins. Of these 6 peptides, only GAP(1–13) partially lowered the plasma levels of PRL at the high dose of 100 μg in OEP rats, but it had no effect on the ether-induced PRL surge at doses of 10 and 100 μg. In conclusion, both GAP(1–13) and GAP(1–23) stimulate FSH and LH release in vivo; these 2 peptides are much less potent in stimulating gonadotropin release than is LHRH. GAP(1–13) exerts a preferential FSH-releasing activity, but its PRL-inhibiting activity is minimal.     

Involvement of dynorphin and the kappa opioid receptor in feeding

Dynorphin-(1–17) produces a highly specific increase in food ingestion. Similar enhancement of food ingestion is found with dynorphin fragments (1–10), (1–11), (1–13) and (3–13) but not with (1–8) and (1–9). Dynorphin B (rimorphin) also enhances food intake. The highly specific kappa agonist U-50,488 also enhances food intake as do a number of other kappa-opiate receptor agonists. These studies provided further support for the role of a highly specific dynorphin-kappa opioid receptor in the modulation of feeding.     

Fourier transform infrared spectroscopy used to evidence the prevention of beta-sheet formation of amyloid beta(1-40) peptide by a short amyloid fragment

Reflectance Fourier transform infrared (FT-IR) microspectroscopy was applied to study the prevention of β-sheet formation of amyloid β (Aβ)(1–40) peptide by co-incubation with a hexapeptide containing a KLVFF sequence (Aβ(15–20) fragment). Second-derivative spectral analysis was used to locate the position of the overlapping components of the amide I band of Aβ peptide and assigned them to different secondary components. The result indicates that each intact sample of Aβ(15–20) fragment or Aβ(1–40) peptide previously incubated in distilled water at 37 °C transformed their secondary structure from 1649 (1651) or 1653 cm⁻¹ to 1624 cm⁻¹, suggesting the transformation from -helix and/or random coil structures to β-sheet structure. By co-incubating both samples with different molar ratio in distilled water at 37 °C, the structural transformation was not found for Aβ(1–40) peptide after 24 h-incubation. But the β-sheet formation of Aβ(1–40) peptide after 48 h-incubation was evidenced from the appearance of the IR peak at 1626 cm⁻¹ by adding a little amount of Aβ(15–20) fragment. There was no β-sheet formation of Aβ(1–40) peptide after addition with much amount of Aβ(15–20) fragment, however, suggesting the higher amount of Aβ(15–20) fragment used might inhibit the β-sheet formation of Aβ(1–40) peptide. The more Aβ(15–20) fragment used made the more stable structure of Aβ(1–40) peptide and the less β-sheet formation of Aβ(1–40) peptide. The study indicates that the reflectance FT-IR microspectroscopy can easily evidence the prevention of β-sheet formation of Aβ(1–40) peptide by a short amyloid fragment.     

Two new coccidia (Apicomplexa:Eimeriidae) from the green peacock (Pavo muticus) from Saudi Arabia

Two new species of Eimeria were found from faecal samples of ten green peacocks (Pavo muticus) collected at Al-Kharj area, a central region of Saudi Arabia. Sporulated oocysts of Eimeria mutica n.sp. are ellipsoidal 23.1×17.4 (22.4–25.0×16.7–18.9) μm, with a smooth bilayered wall. A micropyle and bilobed polar body are present, but without an oocyst residuum. The sporocyst is an elongated-ovoid 13.7×6.2 (12.0–14.2×5.4–6.7) μm, with a Stieda body and a residuum. Sporulated oocysts of E. kharjensis n.sp. are subspherical 20.3×17.7 (19.0–21.5×16.2–18.7) μm, with a two layered wall and a single polar body. The micropyle is covered by a dome-shaped cap and the sporocyst is an elongate-ovoid 12.7×6.3 (11.9–13.5×5.4–6.8) μm, with a Stieda body. The sporocyst residuum is present as several small granules.