Interleukin-1 gene polymorphisms and periodontal status in a Spanish population.

The aim of this study was to investigate the possible association between interleukin (IL)-1A (+4845) and/or IL-1B (+3954) gene polymorphisms and the onset and progression of chronic periodontal disease (PD), an issue that remains controversial. The relationship between IL-1ß concentration in the gingival crevicular fluid (GCF) and disease activity was also evaluated. The study was performed on 25 individuals with no gingivitis or PD and on 25 subjects with active chronic PD. Two samples of GCF were obtained from each subject and IL-1ß was determined by enzyme-linked immunoabsorbent assay. Blood samples (10 ml) were drawn from each subject to detect polymorphisms in IL-1A (+4845) and IL-1B (+3954) by polymerase chain reaction. Mean GCF IL-1ß concentrations were higher in patients with active chronic PD compared to the control group. No significant association was found in either group between GCF IL-1ß concentration and the presence of polymorphisms in IL-1A (+4845), IL-1B (+3954) or both genotypes. No significant difference was found in either group with regard to the presence of polymorphisms in IL-1A (+4845), IL-1B (+3954) or both genotypes (p=0.556). The concentration of IL-1ß in GCF was almost 2-fold higher in patients with chronic PD than in the healthy individuals. The presence of polymorphisms in IL-1A (+4845) and/or IL-1B (+3954) genotypes is not associated with IL-1ß overproduction in GCF and is not a risk factor for chronic PD. IL-1ß is considered a suitable marker of the severity and progression of chronic PD. The presence of IL-1A (+4845) and/or IL-1B +3954 gene polymorphisms does not appear to be a risk factor for chronic PD. Therefore, the IL-1A (+4845) and/or IL-1B +3954 gene polymorphisms cannot be considered genetic markers of chronic PD. Moreover, these polymorphisms do not indicate an overproduction of IL-1ß in GCF.

El polimorfismo de la IL-1 y su eventual asociación con la enfermedad periodontal crónica. Una revisión de la literatura (II) / The association between the IL-1 polymorphism and chronic periodontal disease. A literature review (II)

El propósito de esta revisión es describir el papel que desempeña el polimorfismo de los genes de la IL-1 en la aparición y evolución de la enfermedad periodontal. A lo largo de ella, se observa una falta de homogeneidad en los resultados obtenidos por los diferentes autores. Incluso los estudios más recientes tienden a desestimar la relación que pudiera existir entre la presencia de polimorfismos en los genes de la IL-1 y la enfermedad periodontal. De igual manera, la presencia de estos polimorfismos parece no influir en el fracaso implantario ni en las técnicas de regeneración tisular guiada (AU)

The aim of this bibliographic review is describing the role of the polymorphism in IL-1 genes in the appearance and evolution of the periodontal disease. Through the review, it’s observed a lack of homogeneity in the results obtained by different authors. Despite of this, more recent articles tend to have a low opinion of the relationship that could exist between the presence of polymorphism in IL-1 genes and periodontal disease. In the same way, the presence of theses polymorphisms seems not to influence in the implant failure nor in the guided tissue regeneration techniques (AU)

La IL-1 y su eventual asociación con la enfermedad periodontal crónica. Una revisión de la literatura (I) / The association between IL-1 and chronic periodontal disease. A literature review (I)

El propósito de esta revisión bibliográfica es describir las acciones que protagonizan las interleuquinas, con especial interés la IL-1 en el desarrollo de la enfermedad periodontal crónica, así como su presencia en el fluido gingival crevicular (FGC). La mayoría de los estudios revisados muestran una fuerte asociación entre los niveles aumentados de esta citoquina y el estado periodontal del paciente. La IL-1 puede ser un excelente marcador para detectar la severidad y progresión de la enfermedad periodontal crónica (AU)

The aim of this bibliographic review is describing the actions played by the interleukins, especially IL-1 in development of chronic periodontal disease and it’s presence in gingival crevicular fluid (GCF). Most of the reviewed articles show a strong relationship between high levels of these cytokines and the periodontal status of the patient. Thus, IL-1 becomes in an excellent marker for detecting the severity and progression of chronic periodontal disease (AU)

Induction of the halobenzoate catabolic pathway and cometabolism of ortho-chlorobenzoates in Pseudomonas aeruginosa 142 grown on glucose-supplemented media.

The aerobic cometabolism of ortho-substituted chlorobenzoates by Pseudomonas aeruginosa strain 142 growing on glucose-supplemented medium was analyzed. The strain, which can use 2-chlorobenzoate (2-CBA) and 2,4-dichlorobenzoate (2,4-DCBA) as sole carbon and energy sources, showed high rates of 2-CBA metabolism in glucose-fed cells. In contrast, 2,4-DCBA was metabolized only after extended incubation of the full grown culture and depletion of glucose. In addition to the ortho-dehalogenation (ohb142) genes encoding the alpha and beta subunits of the oxygenase component of a 2-halobenzoate dioxygenase, strain 142 harbours a closely related ohbABCDFG gene cluster previously identified in P. aeruginosa JB2 (ohbJB2). The genes for the chlorocatechol ortho-catabolic pathway were identified and sequenced in this strain, showing a near complete identity with the clcABD operon of the pAC27 plasmid. Relative quantification of mRNA by RT-PCR shows a preferential induction of ohb142 by 2-CBA, which is abolished in glucose-grown cultures. The alternate ohbJB2 and clc genes were expressed preferentially in 2,4-DCBA grown cultures. Only ohbJB2 appears to be expressed in the presence of the carbohydrate. Detection of chlorocatechol-1,2-dioxygenase activity in 2,4-DCBA plus glucose grown cultures suggests the presence of an alternate system for the ortho-cleavage of chlorobenzoates. The recruitment of elements from two halobenzoate dioxygenase systems with different induction patterns, together with a chlorocatechol degradative pathway not repressed by carbon catabolite, may allow P. aeruginosa 142 to cometabolize haloaromatics in carbohydrate grown cultures.

Covalent and metal-chelate immobilization of a modified 2-haloacid dehalogenase for the enzymatic resolution of optically active chloropropionic acid.

The stereospecific L-2-haloacid dehalogenase DehCI from Pseudomonas CBS3 was tagged with a peptide tail containing six histidines and overexpressed in Escherichia coli. The His-tagged protein was purified after a single-step affinity chromatography on Zn(2+)-chelating sepharose. The activity of the modified protein was tested after immobilization on Zn(2+)-chelating sepharose and on covalently bound acrylic polymer. Both immobilization systems were used for the transformation of racemic 2-chloropropionic acid into D-lactate and D-chloropropionic acid. Although immobilization on chelating sepharose produced a limited increase in stability, covalent immobilization on acrylic polymer significantly extended the operational temperature and pH range of the enzyme: up to 60% of activity was recovered at either 80 degrees C or pH 11, whereas no activity could be detected under these conditions in the soluble or chelate-immobilized enzyme. Both forms of immobilization extended the enzyme effective storage periods, and after 10 cycles of reutilization, 70% and 20% of the initial activity was recovered in the covalent- and chelate-immobilized enzyme, respectively.

Structural defects underlying protein dysfunction in human glucose-6-phosphate dehydrogenase A(-) deficiency.

The enzyme variant glucose-6-phosphate dehydrogenase (G6PD) A(-), which gives rise to human glucose-6-phosphate dehydrogenase deficiency, is a protein of markedly reduced structural stability. This variant differs from the normal enzyme, G6PD B, in two amino acid substitutions. A further nondeficient variant, G6PD A, bears only one of these two mutations and is structurally stable. In this study, the synergistic structural defect in recombinant G6PD A(-) was reflected by reduced unfolding enthalpy due to loss of beta-sheet and alpha-helix interactions where both mutations are found. This was accompanied by changes in inner spatial distances between residues in the coenzyme domain and the partial disruption of tertiary structure with no significant loss of secondary structure. However, the secondary structure of G6PD A(-) was qualitatively affected by an increase in beta-sheets substituting beta-turns related to the lower unfolding enthalpy. The structural changes observed did not affect the active site of the mutant proteins, since its spatial position was unmodified. The final result is a loss of folding determinants leading to a protein with decreased intracellular stability. This is suggested as the cause of the enzyme deficiency in the red blood cell, which is unable to perform de novo protein synthesis.

Molecular phylogeny and morphological homoplasy in fruitbats.

The present study evaluates the evolutionary framework of the Old World fruitbats based on the cytochrome b and 16S rRNA mitochondrial gene sequences from a wide range of taxa. Phylogenetic analyses indicated that morphology-based subfamilies and most suprageneric groups are nonnatural assemblages. They also support the existence of an endemic African clade of fruitbats. The discrepancy between the evolutionary relationships yielded by molecular and morphological data sets may be, at least in part, explained by the recurrent retention of primitive morphology (Rousettus-like) across different lineages. The maintenance of primitive characters in different groups of flying foxes, as well as morphological convergence in nectar-feeding bats and possibly also in short-muzzle bats, may have led to high levels of homoplasy, resulting in misleading taxonomic arrangements. This may be particularly so with respect to high taxonomic levels based on morphological characters.

Phylogeography of African fruitbats (Megachiroptera).

Joint sequences from the mitochondrial cytochrome b and 16S rRNA genes of a wide representation of Megachiroptera were employed to evaluate the traditional taxonomic arrangement of African fruitbats and to examine their origins and evolutionary relationships. The resulting phylogenetic hypotheses are inconsistent with the previously established morphology-based subdivisions of Megachiroptera at the suprageneric level. Findings indicate the existence of an African clade, which appears to be formed by two endemic clades: the epomophorines and the myonycterines. According to our topologies, the genus Rousettus is monospecific in mainland Africa. Its traditional subgenera Stenonycteris and Lissonycteris appear closer to the myonycterines than to Rousettus. Topologies also indicate that the African genus Eidolon is not phylogenetically related to any other African fruitbat. It would seem that the arrival of fruitbats in Africa was a complex process involving at least three independent colonization events. One event took place probably in the Miocene via forested corridors that connected the African and Asian rain forest blocks, as for other groups of mammals. The resulting lineage diversified into most of the extant African fruitbats. Related to this clade, the Rousettus species group is thought to have arrived in Africa in more recent times, possibly by progressive displacement from the East through India. Finally, the present topologies suggest an independent colonization of Africa by ancestors of Eidolon.

Unproductive folding of the human G6PD-deficient variant A-.

Human glucose-6-phosphate dehydrogenase (G6PD) deficiency almost invariably results from the presence of missense mutations in the X-linked gene encoding G6PD. The common African deficient variant G6PD A- differs from the normal G6PD B by two amino acid substitutions. Only one of these mutations is found on its own, resulting in the nondeficient variant G6PD A. Deficiency is always associated with decreased G6PD activity in red cells, leading to a variety of clinical manifestations. A group of deficient variants, including A-, have near-normal affinity for the substrates G6P and NADP. In these cases, deficiency is caused by a decreased number of catalytically active molecules per cell due to intracellular instability of the mutated G6PD, although the mechanism for this in vivo instability is unknown. Here we report that in vitro folding of the A- variant mainly renders partially folded polypeptides that do not undergo the dimerization required for activity. Under the same conditions, the nondeficient variants B and A undergo folding to produce active dimers with normal mobilities in native gels and normal kinetic properties. The loss of intrinsic folding determinants in the A- variant may underlie the mechanism of its in vivo instability.

The complete nucleotide sequence of the mitochondrial DNA genome of the rainbow trout, Oncorhynchus mykiss.

The complete nucleotide sequence of the mitochondrial DNA of the rainbow trout, Onchorynchus mykiss, has been determined. The total length of the molecule is 16,660 bp. The rainbow trout mitochondrial DNA has the same organization described in eutherian mammals, the clawed frog (Xenopus laevis), and the two fish species, Oriental stream loach (Crossotoma lacustre) and carp (Cyprinus carpio). Alignment and comparison of the deduced amino acid sequences of the 13 proteins encoded by rainbow trout and other vertebrate mitochondrial genomes allowed us to estimate that COI is the most conserved mitochondrial subunit (amino acid identity ranging from 85.6% to 94.8%) whereas ATPase 8 is the most variable one (amino acid identity ranging from 30.8% to 70.4%). Putative secondary structures for the 22 tRNAs found in the molecule are given along with an extensive comparison of tRNA sequences among representative species of each major group of vertebrates. In this sense, an unusual cloverleaf structure for the tRNASer(AGY) is proposed. A stem-loop structure inferred for the origin of the L-strand replication (OL) and the presence of a large polycytidine tract in the OL loop is described. The existence of this stretch instead of the usual T-rich sequence reported so far in mammal mtDNAs is explained in terms of a less-strict template dependence of the RNA primase involved in the initiation of L-strand replication.

Revised dinoflagellate phylogeny inferred from molecular analysis of large-subunit ribosomal RNA gene sequences.

The nucleotide sequence analysis of the PCR products corresponding to the variable large-subunit rRNA domains D1, D2, D9, and D10 from ten representative dinoflagellate species is reported. Species were selected among the main laboratory-grown dinoflagellate groups: Prorocentrales, Gymnodiniales, and Peridiniales which comprise a variety of morphological and ecological characteristics. The sequence alignments comprising up to 1,000 nucleotides from all ten species were employed to analyze the phylogenetic relationships among these dinoflagellates. Maximum parsimony and neighbor-joining trees were inferred from the data generated and subsequently tested by bootstrapping. Both the D1/D2 and the D9/D10 regions led to coherent trees in which the main class of dinoflagellates. Dinophyceae, is divided in three groups: prorocentroid, gymnodinioid, and peridinioid. An interesting outcome from the molecular phylogeny obtained was the uncertain emergence of Prorocentrum lima. The molecular results reported agreed with morphological classifications within Peridiniales but not with those of Prorocentrales and Gymnodiniales. Additionally, the sequence comparison analysis provided strong evidence to suggest that Alexandrium minutum and Alexandrium lusitanicum were synonymous species given the identical sequence they shared. Moreover, clone Gg1V, which was determined Gymnodinium catenatum based on morphological criteria, would correspond to a new species of the genus Gymnodinium as its sequence clearly differed from that obtained in G. catenatum. The sequence of the amplified fragments was demonstrated to be a valuable tool for phylogenetic and taxonomical analysis among these highly diversified species.

Purification and characterization of 4-hydroxybenzoate 3-hydroxylase from a Klebsiella pneumoniae mutant strain.

Unlike the parent wild-type strain, the Klebsiella pneumoniae mutant strain MAO4 has a 4-HBA+ phenotype. The capacity of this mutant to take up and metabolize 4-hydroxybenzoate (4-HBA) relies on the expression of a permease and an NADPH-linked monooxygenase (4-HBA-3-hydroxylase). Both enzymes are normally expressed at basal levels, and only the presence of 4-HBA in the media enhances their activities. Strikingly, when the Acinetobacter calcoaceticus pobA gene encoding 4-hydroxybenzoate-3-hydroxylase was expressed in hydroxybenzoate K. pneumoniae wild-type, the bacteria were unable to grow on 4-HBA, suggesting that the main difference between the wild-type and the mutant strain is the capability of the latter to take up 4-HBA. 4-HBA-3-hydroxylase was purified to homogeneity by affinity, gel-filtration, and anion-exchange chromatography. The native enzyme, which appeared to be a dimer of identical subunits, had an apparent molecular mass of 80 kDa and a pI of 4.6. Steady-state kinetics were analyzed; the initial velocity patterns were consistent with a concerted substitution mechanism. The purified enzyme had 362 amino acid residues, and a tyrosine seemed to be involved in substrate activation.

Nucleotide sequence of the sheep mitochondrial DNA D-loop and its flanking tRNA genes.

The nucleotide sequence of the sheep mitochondrial DNA displacement-loop (D-loop) region and its flanking tRNA genes has been determined. Several conserved motifs among mammals have been identified along the 1189-bp sequence of the sheep control region: ten termination-associated sequences (TASs) and one conserved sequence block (CSB-1). CSB-2 and CSB-3, which are frequently found in most species, are not present in the sheep D-loop, which shows instead a short direct repeat at their usual localization. A long polypyrimidine tract between CSB-1 and the tRNA(Phe) gene is also present. Furthermore, the sheep mtDNA D-loop region displays tandem repeats in the left domain (adjacent to the tRNA(Pro) gene) comprising three different termination-associated sequences (TAS-5, TAS-6 and TAS-7).

Purification and characterization of the 3-hydroxybenzoate-6-hydroxylase from Klebsiella pneumoniae.

We isolated 3-hydroxybenzoate-6-hydroxylase (E.C.1.14.13.), an inducible enzyme that catalyzed the para-hydroxylation of 3-hydroxybenzoate (3-HBA) to 2,5-dihydroxybenzoate, from Klebsiella pneumoniae. Although the enzyme was found to be mainly induced by its substrate, a coordinated induction of 3-hydroxybenzoate hydroxylase and gentisate dioxygenase was also observed in the presence of the product of the reaction. The purified enzyme was a monomer with a molecular mass of 42,000. It contained FAD as a prosthetic group, utilized NADH or NADPH with similar efficiencies and its activity was inhibited by Cu2+, Fe2+ and Hg2+. Other properties, such as induction mechanism and kinetic parameters were also studied. Moreover, for the first time the amino acid composition of a 3-hydroxybenzoate-6-hydroxylase was determined.

Lipogenic activities in rat liver are subjected to circadian rhythms.

Diurnal variations were investigated in the activities of the key lipogenic enzymes in rat liver under standard experimental conditions. Fatty acid synthetase, acetyl-CoA-carboxylase, and ATP-citrate lyase showed sinusoidal circadian rhythms (statistically sustained), reaching maximum activity at night and minimum during the light period, with a fold increase value of 1.7, 2.2 and 3.2, respectively. Although a non-sinusoidal circadian rhythm was observed in the malic enzyme activity, statistically different time-dependent activity values were detected throughout a day. The observed rhythms run in parallel with that previously reported for glucose-6-phosphate dehydrogenase, suggesting a whole circadian regulation for rat liver lipogenesis.

3,4-Dihydroxyphenylacetate 2,3-dioxygenase from Klebsiella pneumoniae, a Mg(2+)-containing dioxygenase involved in aromatic catabolism.

3,4-Dihydroxyphenylacetate 2,3-dioxygenase, an extradiol-ring-cleavage dioxygenase, has been purified from Klebsiella pneumoniae to homogeneity. The enzyme has an M(r) of 102,000 in its tetrameric form with an M(r) of 25,500 for each subunit. Unlike most other dioxygenases, the enzyme reported here contains Mg2+, as determined by atomic-absorption spectrophotometry and plasma emission metal analysis. The enzyme was shown to contain approx. 1 g-atom of Mg2+/mol of protein and we suggest an alpha 4 Mg2+ quaternary structure. This is the first report of a dioxygenase containing Mg2+ in its structure.

High resolution of proteins by double-inverted gradient polyacrylamide gel electrophoresis (DG-PAGE).

We have designed a new method for high resolution electrophoretic separation of proteins that have similar molecular weights. The proteins migrate first through a conventional gradient gel, in which molecular friction increases as pore size decreases. The proteins then enter an inverted sodium dodecyl sulfate (SDS) gradient gel in which friction decreases; thus, smaller molecules gradually migrate faster and achieve improved separation from larger molecules, which remain near the border between the two gels. We therefore call this technique double-inverted gradient polyacrylamide gel electrophoresis (DG-PAGE). This technique was used to resolve mixtures of aldolase, horseradish peroxidase precursors, glucose 6-phosphate dehydrogenase and pyruvate kinase. By comparison with other established methods, we show that DG-PAGE has a higher resolving power, which achieves clear separation of proteins differing as little as 0.5 kDa in molecular weight.

4-Hydroxybenzoate uptake in Klebsiella pneumoniae is driven by electrical potential.

The uptake of 4-hydroxybenzoic acid (4-HBA) in intact cells of a mutant of Klebsiella pneumoniae was investigated. Uptake of 4-HBA was shown to be an inducible system. This uptake system, at pH 7.0, has a high affinity for its substrate (apparent Kt = 13 microM) and a maximal velocity of 27.6 nmol min-1 mg protein-1. Competition studies with various structural analogs indicated a very narrow specificity of the 4-HBA uptake system. The transport system has been inhibited by inhibitors of energy metabolism and its activity has not been detected in the crude shock extracts. The effect of two ionophores, nigericin and valinomycin, on 4-HBA uptake with respect to the external pH has been studied. All observations indicate that 4-HBA uptake is active and energized by the membrane potential.

Transport of 4-hydroxyphenylacetic acid in Klebsiella pneumoniae.

Klebsiella pneumoniae M5a1 has been shown to possess an inducible transport system for 4-hydroxyphenylacetate (4-HPA). This transport system has a Kt of 16.3 microM and a maximal velocity of 31.2 nmol/min (milligrams dry weight). The transport system has been inhibited by inhibitors of energy metabolism with a concomitant decrease in cellular ATP concentrations, and the 4-HPA binding activity has been detected in the crude shock extracts. All these observations indicate that 4-HPA uptake is an active transport which involves a periplasmic binding protein and it seems to be energized by phosphate bond energy.