Comparative analysis of seven multiple protein sequence alignment servers: clues to enhance reliability of predictions.

MOTIVATION: The prediction reliability of seven multiple alignment servers currently available on the Internet (ClustalW, MAP, PIMA, Block Maker, MSA, MEME and Match-Box) has been evaluated in terms of power (sensitivity) and confidence (selectivity). Therefore, the alignments obtained have been respectively compared to refined structural alignments for 20 families of related proteins with low levels of identity. RESULTS: Results clearly show that any powerful method remains reliable when the rate of identity falls. For some methods, power and confidence decrease linearly with the rate of identity, while other methods emphasize reliability at the cost of a lower power. Increasing the number of related sequences included in the alignment may either improve or decrease the quality of the predictions substantially. For some methods, the gain in power or in confidence is quite systematic; for others, the effect of the addition of homologous sequences is highly unpredictable. Extracting the consensus between two different methods may increase the overall confidence of the predictions tremendously. Our conclusions induce users of sequence alignment methods on the Internet to select the most suitable technique according to their requirements in terms of selectivity and sensitivity. AVAILABILITY: The aligned sequences of the 20 alignments of structure can be obtained automatically by sending the message 'send: cabios_tests.txt' by e-mail to 'matchbox@biq.fundp.ac.be'. CONTACT: eric.depiereux@fundp.ac.be

Match-Box_server: a multiple sequence alignment tool placing emphasis on reliability.

MOTIVATION: The Match-Box software comprises protein sequence alignment tools based on strict statistical thresholds of similarity between protein segments. The method circumvents the gap penalty requirement: gaps being the result of the alignment and not a governing parameter of the procedure. The reliable conserved regions outlined by Match-Box are particularly relevant for homology modelling of protein structures, prediction of essential residues for site-directed mutagenesis and oligonucleotide design for cloning homologous genes by polymerase chain reaction (PCR). RESULTS: The method produces reliable results, as assessed by tests performed on protein families of known structures and of low sequence similarity. A reliability score is computed in relation to a threshold of similarity progressively raised to extend the aligned regions to their maximal length, up to the significance limit of matching segments. The score obtained at each position is printed below the sequences and allows a discriminant reading of each aligned region. AVAILABILITY: Sequences may be submitted to a Web server at http://www.fundp.ac.be/sciences/biologie/bms/+ ++matchbox_submit.html or sent by e-mail to matchbox/biq.fundp.ac.be (help available by just mailing help).

A lack of direct action of glucagon on kidney metabolism, hemodynamics, and renal sodium handling in the dog.

Although several reports suggest that pharmacologic amounts of glucagon may promote natriuresis, the influence of a physiological or even pathophysiological increase in circulating glucagon levels on kidney function has never been convincingly demonstrated. The present study was therefore undertaken to determine whether a moderate increase in plasma glucagon concentration of blood perfusing the kidney may influence kidney function and promote urinary sodium excretion. To this end, glucagon was infused directly into one renal artery of anesthetized dogs at a rate of 1 ng x kg(-1) x min(-1), calculated to increase glucagon concentration in the blood perfusing the kidney within the pathophysiologic range and thus to levels seen in some catabolic states such as poorly controlled diabetes or starvation. The contralateral kidney was infused with saline only. The estimated concentration of glucagon in blood perfusing the hormone-infused kidney increased with glucagon infusion from 227 pg x mL(-1) during the control period to mean of 779 pg x mL(-1). There was a significant increase in glucagon extraction by this kidney, from 33% in baseline conditions to 61% upon intrarenal infusion of the hormone, and hence venous glucagon levels were only slightly higher than in the contralateral kidney. Despite a more than threefold increase in glucagon levels in blood perfusing the hormone-infused kidney versus the contralateral kidney, this experimentally induced hyperglucagonemia was without influence on renal plasma flow (RFP), glomerular filtration rate (GFR), renal vascular resistance, renal uptake of oxygen and energy-providing substrates. Excretion of Na+, K+, Cl-, and PO4(3-) was likewise unaffected. These results indicate that hyperglucagonemia, at least of a magnitude comparable to that seen in starvation or diabetic decompensation, is devoid of any detectable direct influence on renal hemodynamics or tubular function.

Cloning and sequencing of the bacterioferritin gene of Brucella melitensis 16M strain.

The 40 N-terminal amino acids of the 20 kDa antigen A2 from Brucella melitensis were sequenced and showed important similarities with 4 bacterioferrins. A monoclonal antibody raised against this antigen cross-reacted with Escherichia coli bacterioferritin. Hybridization of two sets of degenerate primers with B. melitensis HindIII-digested genomic DNA identified a 3.8 kb fragment. This fragment was shown to contain a bacterioferritin gene (bfr) encoding a 161-amino acid protein. The sequence of the Brucella bacterioferritin is 69% similar to that of E. coli, and many of the ferroxidase centre and haem-ligation residues are conserved.

Ciliation of bovine aortic endothelial cells in culture.

Solitary cilia were observed by electron microscopy in senescent bovine aortic endothelial cells in culture. Such single cilia have previously been seen in several tissues of various species, but as far as we know they have not been identified in cultured endothelial cells. The analysis of ultrathin sections enabled us to show that the cilia originated in one of the two centrioles. Vacuolar structures located at one end of the centrioles were also observed and might occur during the lengthening of the cilium. Moreover, the surface replication technique allowed us to show that the cilia could extend out of the cell. As senescent endothelial cells enter a quiescent stage, they could build up such a cilium as was observed for some strains of cultured fibroblasts.

Increased ketone utilization by the kidney reduces renal lactate uptake but does not affect tubular sodium reabsorption.

The purpose of the present study was to assess whether a moderate increase in ketonemia interferes with renal uptake of energy-providing substrates, and whether it may promote natriuresis similar to that occurring spontaneously during the early phase of starvation. To this end, the sodium salt of D(-)3-hydroxybutyrate (3OHB) was infused to 10 anesthetized dogs at a rate of 20 mumol/kg.min-1 over 135 minutes. To allow comparison, an equivalent amount of sodium was infused as sodium bicarbonate to 10 other dogs (to induce an alkalinization similar to that resulting from the utilization of the sodium salt of 3OHB), while 10 additional dogs received an equimolar infusion of NaCl to provide reference values. Before 3OHB or bicarbonate infusion, lactate represented the major fuel taken up by the kidney (28 +/- 3 mumol/100 g kidney.min-1 on average). While small but significant amounts of 3OHB were taken up by the kidney in control conditions (0.7 +/- 0.1 mumol/100 g.min-1; P < .05), there was no significant uptake of free fatty acids (FFA) or glucose. Upon 3OHB infusion, which increased plasma 3OHB levels to 1.7 mmol/L, the renal uptake of this substrate increased to 25 +/- 3 mumol/100 g.min-1, in proportion to renal 3OHB availability (r = .73, P < .001). Despite an increase in arterial lactate levels that occurred during 3OHB infusion, renal lactate uptake and the extraction ratio of this substrate were decreased (P < .001).(ABSTRACT TRUNCATED AT 250 WORDS)

Reappraisal of the role of insulin on sodium handling by the kidney: f1fect of intrarenal insulin infusion in the dog.

Since several studies suggest that increased insulin levels may induce antinatriuresis, the present work was undertaken to determine whether a physiological increase in insulin levels in blood perfusing the kidney may exert direct effect on kidney function, and more specifically on sodium reabsorption. To this end, insulin was infused directly into one renal artery of 10 anaesthetized dogs at the rate of 4 mU min-1 for a period of 90 min. The contralateral kidney was infused with saline alone, to provide reference values. Insulin level in the renal vein of the insulin-infused kidney went up from 1.4 +/- 0.9 before to 30.6 +/- 7.1 microU ml-1 after 90 min of insulin perfusion. There was no significant effect on renal plasma flow, glomerular filtration rate and renal uptake of substrates or oxygen between ipsi- and contralateral kidney. The fractional excretion of sodium was likewise unaffected, since it averaged at the end of insulin infusion 0.41 +/- 0.11% vs. 0.50 +/- 0.14% for the contralateral saline infused kidney. Even if one may assume that the baseline low insulin concentrations promote tubular sodium reabsorption, the results of the present study suggest that a moderate hyperinsulinaemia is without any additional effect on renal sodium handling.