Cloning and characterization of a new laccase from Lactobacillus plantarum J16 CECT 8944 catalyzing biogenic amines degradation.

In our search for degrading activities of biogenic amines (BAs) in lactic acid bacteria, a protein annotated as laccase enzyme was identified in Lactobacillus plantarum J16 (CECT 8944). In this study, the gene of this new laccase was cloned and heterologously overexpressed in Escherichia coli. The recombinant laccase protein was purified and characterized biochemically. The purified laccase showed characteristic spectroscopic properties of blue multicopper oxidases. The enzyme has a molecular weight of ∼ 62.5 kDa and activity toward typical laccase substrates 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,6-dimethoxyphenol (2,6-DMP). The pH optima on ABTS and 2,6-DMP were 3.5 and 7.0, respectively. Kinetic constants Km and Vmax were of 0.21 mM and 0.54 U/mg for ABTS and 1.67 mM and 0.095 U/mg for 2,6-DMP, respectively. The highest oxidizing activity toward 2,6-DMP was obtained at 60 °C. However, after a preincubation step at 85 °C for 10 min, no residual activity was detected. It has been demonstrated that recombinant L. plantarum laccase oxidizes biogenic amines, mainly tyramine, and thus presents new biotechnological potential for the enzyme in eliminating toxic compounds present in fermented food and beverages.

Identification of a novel enzymatic activity from lactic acid bacteria able to degrade biogenic amines in wine.

The main objectives of this study were the search for enzymatic activities responsible for biogenic amine (BA) degradation in lactic acid bacteria (LAB) strains isolated from wine, their identification, and the evaluation of their applicability for reducing BAs in wine. Fifty-three percent of the 76 LAB cell extracts showed activity against a mixture of histamine, tyramine, and putrescine when analyzed in-gel. The quantification of the degrading ability for each individual amine was tested in a synthetic medium and wine. Most of the bacteria analyzed were able to degrade the three amines in both conditions. The highest percentages of degradation in wine were those of putrescine: up to 41% diminution in 1 week. Enzymes responsible for amine degradation were isolated and purified from Lactobacillus plantarum J16 and Pediococcus acidilactici CECT 5930 strains and were identified as multicopper oxidases. This is the first report of an efficient BA reduction in wine by LAB. Furthermore, the identity of the enzymes involved has been revealed.

Mononucleosis infecciosa y trombopenia grave / Infectious mononucleosis and severe thrombocytopenia

La mononucleosis infecciosa por el virus de Epstein-Barr es habitualmente una enfermedad benigna y autolimitada. Se presentan 2 casos clínicos ingresados en la misma semana, con sospecha de coinfección por otros virus (citomegalovirus y virus herpes humano 6), que presentaron trombopenia grave en la fase aguda de la enfermedad. La evolución y el tratamiento fueron diferentes en cada caso(AU)

Infectious mononucleosis by Epstein-Barr virus is usually a benign self-limiting disease. We present two cases admitted in the same week, with suspected co-infection by other viruses (cytomegalovirus and human herpes virus 6), which showed severe thrombocytopenia in the acute phase of the disease. The progress and treatment were different in each case(AU)

[Infectious mononucleosis and severe thrombocytopenia]. / Mononucleosis infecciosa y trombopenia grave.

Infectious mononucleosis by Epstein-Barr virus is usually a benign self-limiting disease. We present two cases admitted in the same week, with suspected co-infection by other viruses (cytomegalovirus and human herpes virus 6), which showed severe thrombocytopenia in the acute phase of the disease. The progress and treatment were different in each case.

Bromodomain factor 1 (Bdf1) protein interacts with histones.

Using a yeast two-hybrid assay we detected an interaction between the N-terminal region of histone H4 (amino acids 1--59) and a fragment of the bromodomain factor 1 protein (Bdf1p) (amino acids 304--571) that includes one of the two bromodomains of this protein. No interaction was observed using fragments of histone H4 sequence smaller than the first 59 amino acids. Recombinant Bdf1p (rBdf1p) demonstrates binding affinity for histones H4 and H3 but not H2A and H2B in vitro. Moreover, rBdf1p is able to bind histones H3 and H4 having different degrees of acetylation. Finally, we have not detected histone acetyltransferase activity associated with Bdf1p.

The yeast histone acetyltransferase A2 complex, but not free Gcn5p, binds stably to nucleosomal arrays.

We have investigated the structural basis for the differential catalytic function of the yeast Gcn5p-containing histone acetyltransferase (HAT) A2 complex and free recombinant yeast Gcn5p (rGcn5p). HAT A2 is shown to be a unique complex that contains Gcn5p, Ada2p, and Ada3p, but not proteins specific to other related HAT A complexes, e.g. ADA, SAGA. Nevertheless, HAT A2 produces the same unique polyacetylation pattern of nucleosomal substrates reported previously for ADA and SAGA, demonstrating that proteins specific to the ADA and SAGA complexes do not influence the enzymatic activity of Gcn5p within the HAT A2 complex. To investigate the role of substrate interactions in the differential behavior of free and complexed Gcn5p, sucrose density gradient centrifugation was used to characterize the binding of HAT A2 and free rGcn5p to intact and trypsinized nucleosomal arrays, H3/H4 tetramer arrays, and nucleosome core particles. We find that HAT A2 forms stable complexes with all nucleosomal substrates tested. In distinct contrast, rGcn5p does not interact stably with nucleosomal arrays, despite being able to specifically monoacetylate the H3 N terminus of nucleosomal substrates. Our data suggest that the ability of the HAT A2 complex to bind stably to nucleosomal arrays is functionally related to both local and global acetylation by the complexed and free forms of Gcn5p.

RPD3-type histone deacetylases in maize embryos.

Posttranslational core histone acetylation is established and maintained by histone acetyltransferases and deacetylases. Both have been identified as important transcriptional regulators in various eukaryotic systems. In contrast to nonplant systems where only RPD3-related histone deacetylases (HD) have been characterized so far, maize embryos contain three unrelated families of deacetylases (HD1A, HD1B, and HD2). Purification, cDNA cloning, and immunological studies identified the two maize histone deacetylase HD1B forms as close homologues of the RPD3-type deacetylase HDAC1. Unlike the other maize deacetylases, HD1A and nucleolar HD2, HD1B copurified as a complex with a protein related to the retinoblastoma-associated protein, Rbap46. Two HD1B mRNA species could be detected on RNA blots, encoding proteins of 58 kDa (HD1B-I) and 51 kDa (HD1B-II). HD1B-I (zmRpd3) represents the major enzyme form as judged from RNA and immunoblots. Levels of expression of HD1B-I and -II mRNA differ during early embryo germination; HD1B-I mRNA and protein are present during the entire germination pathway, even in the quiescent embryo, whereas HD1B-II expression starts when meristematic cells enter S-phase of the cell cycle. In line with previous results, HD1B exists as soluble and chromatin-bound enzyme forms. In vivo treatment of meristematic tissue with the deacetylase inhibitor HC toxin does not affect the expression of the three maize histone deacetylases, whereas it causes downregulation of histone acetyltransferase B.

Mobility of acetylated histones in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

We describe an altered mobility for acetylated histone isoforms in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoforms of histones H3 and H4 with a higher acetylation degree have a slightly faster electrophoretic mobility. Since acetylation neutralizes the positive charge of the epsilon-amino group of lysine, without significantly changing the molecular mass of the protein, the acetylation-dependent mobility shift could be explained by the increase of the net negative charge of the SDS-histone complexes. A possible consequence of this differential mobility for the acetylation site determination by protein microsequencing from SDS gels is discussed.

[Improvement in fibrinolytic function following anticoagulant treatment in chronic rheumatic atrial fibrillation]. / Mejoría de la función fibrinolítica tras el tratamiento anticoagulante en la fibrilación auricular reumática crónica.

INTRODUCTION AND OBJECTIVES: Patients with rheumatic atrial fibrillation are considered at high risk of systemic embolism and require oral anticoagulation. Fibrinolytic function has been little studied. We evaluated fibrinolytic activation markers before starting anticoagulation, at 1 and 6 months following the introduction of oral anticoagulation therapy. We analyzed the relationship with left atrial diameter and mitral area. METHODS: Tissue plasminogen activator (tPA), its inhibitor (PAI-1), plasmin-antiplasmin complexes (PAP) and D-dimer were measured in 13 patients with rheumatic atrial fibrillation. Basal levels were compared with those found in plasma of 20 healthy subjects matched by sex and age. Transthoracic echocardiography was made. RESULTS: A significant increase for PAI-1 and D-dimer levels were detected in patients with atrial fibrillation group (p < 0.05), with no differences in tPA and PAP concentrations. Significant correlation between left atrial diameter and basal t-PA levels was found. Levels of t-PA, PAI-1 and D-dimer decreased significantly under anticoagulation therapy, whereas PAP levels were significantly increased. CONCLUSIONS: Patients with rheumatic atrial fibrillation show a relative hypofibrinolytic state due to elevated PAI-1 levels with no increase in PAP concentration. At six months of anticoagulation therapy, an improvement of fibrinolytic function markers was observed. This is consistent with the prophylactic effect of oral anticoagulants therapy against thromboembolic risk.

Gcn5p, a transcription-related histone acetyltransferase, acetylates nucleosomes and folded nucleosomal arrays in the absence of other protein subunits.

Gcn5p is the catalytic subunit of several type A histone acetyltransferases (HATs). Previous studies performed under a limited range of solution conditions have found that nucleosome core particles and nucleosomal arrays can be acetylated by Gcn5p only when it is complexed with other proteins, e.g. Gcn5-Ada, HAT-A2, and SAGA. Here we demonstrate that when assayed in buffer containing optimum concentrations of either NaCl or MgCl2, purified yeast recombinant Gcn5p (rGcn5p) efficiently acetylates both nucleosome core particles and nucleosomal arrays. Furthermore, under conditions where nucleosomal arrays are extensively folded, rGcn5p acetylates folded arrays approximately 40% faster than nucleosome core particles. Finally, rGcn5p polyacetylates the N termini of free histone H3 but only monoacetylates H3 in nucleosomes and nucleosomal arrays. These results demonstrate both that rGcn5p in and of itself is catalytically active when assayed under optimal solution conditions and that this enzyme prefers folded nucleosomal arrays as a substrate. They further suggest that the structure of the histone H3 N terminus, and concomitantly the accessibility of the H3 acetylation sites, changes upon assembly into nucleosomes and nucleosomal arrays.

HAT1 and HAT2 proteins are components of a yeast nuclear histone acetyltransferase enzyme specific for free histone H4.

We have analyzed the histone acetyltransferase enzymes obtained from a series of yeast hat1, hat2, and gcn5 single mutants and hat1,hat2 and hat1,gcn5 double mutants. Extracts prepared from both hat1 and hat2 mutant strains specifically lack the following two histone acetyltransferase activities: the well known cytoplasmic type B enzyme and a free histone H4-specific histone acetyltransferase located in the nucleus. The catalytic subunits of both cytoplasmic and nuclear enzymes have identical molecular masses (42 kDa), the same as that of HAT1. However, the cytoplasmic complex has a molecular mass (150 kDa) greater than that of the nuclear complex (110 kDa). The possible functions of HAT1 and HAT2 in the yeast nucleus are discussed. In addition, we have detected a yeast histone acetyltransferase not previously described, designated HAT-A4. This enzyme is located in the nucleus and is able to acetylate free and nucleosome-bound histones H3 and H4. Finally, we show that the hat1, gcn5 double mutant is viable and does not exhibit a new phenotype, thus suggesting the existence of several histone acetyltransferases with overlapping functions.

Interaction between N-terminal domain of H4 and DNA is regulated by the acetylation degree.

To study whether the acetylation of one or more of the four acetylatable lysines of histone H4 affects its binding to DNA, we have designed a protection experiment with a model system consisting in phage lambda DNA as substrate, StuI as restriction endonuclease and histone H4 with different degrees of acetylation as the protective agent. It can be deduced from the experimental data that the protection afforded by the histone is not dependent on the number of positive charges lost by acetylation. Thus, non-acetylated H4 and mono-acetylated H4 cause similar protection, while di-acetylation of the histone seems to be the crucial step in significantly weakening the interaction between H4 and DNA. This is confirmed by the results obtained in protection experiments carried out using H4 peptide (1-24) with different degrees of acetylation as the protecting agent. As restriction enzyme can imitate any trans-acting factor with sequence recognition, the di-acetylated isoform of histone H4 can be the starting point, through acetylation, to unmask DNA sequences, allowing the accessibility of regulatory factors to DNA in the chromatin.

Gcn5p is involved in the acetylation of histone H3 in nucleosomes.

Enzymatic extracts from a gcn5 mutant and wild-type strains of Saccharomyces cerevisiae were chromatographically fractionated and the histone acetyltransferase activities compared. When free histones were used as substrate, extracts from wild-type cells showed two peaks of activity on histone H3 but extracts from gcn5 mutant cells showed only one. With nucleosomes as substrate, the histone acetyltransferase activities present in extracts from the gcn5 mutant strain were not able to modify H3 whereas wild-type cell extracts acetylated intensely this histone. The activity that acetylated nucleosome-bound H3 behaved as a 170-kDa complex. We suggest that Gcn5p represents a catalytic subunit within a multiprotein complex containing proteins that confer on it the ability to acetylate H3 in nucleosomes.

Enzyme-linked immunosorbent assay for detection of Aeromonas hydrophila serogroup O:19.

An enzyme-linked immunosorbent assay has been developed for the detection of Aeromonas hydrophila serogroup O:19 isolated from epizootics in eels. The enzyme-linked immunosorbent assay specificity was confirmed after testing A. hydrophila O:19 and non-O:19 strains from different origins, as well as other Aeromonas species and other fish pathogens such as Vibrio vulnificus biotype 2, V. furnisii, V. damsela, Yersinia ruckerii and Edwardsiella tarda. The detection limits for A. hydrophila O:19 cells were around 10(4)-10(5) cells/well. Artificially infected eels were analyzed and the immunodetection was confirmed by cultural methods. With this methodology A. hydrophila O:19 was successfully detected in infected eels and water samples. We described two subgroups within the serogroup O:19 (Guinée and Jansen system), one of them presents a 50 kDa outer membrane protein as a strong thermostable antigen which is not present in the other group.

Subcellular location of enzymes involved in core histone acetylation.

Multiple enzyme forms of histone deacetylase and histone acetyltransferase exist in germinating maize embryos. We analyzed the association of the different enzymes to chromatin by ion exchange chromatography of subcellular fractions from different time points of embryo germination. The vast majority of histone deacetylase HD-1A was not bound to chromatin, since it was solubilized during chromatin isolation, regardless of its phosphorylation state and the phase of embryo germination. In contrast, HD-2 was chromatin bound during the entire germination pathway. Histone deacetylase HD-1B was present in a chromatin-bound and a soluble form; the ratio between these two forms changed during germination. Both nuclear histone acetyltransferases, HAT-A1 and HAT-A2, were tightly chromatin-bound and could only be released from chromatin by salt extraction. To test whether histone acetyltransferases or deacetylases are associated with the nuclear matrix, we analyzed nuclear matrix preparations from yeast, Physarum, and maize step by step for both enzyme activities. This analysis confirmed that part of the activity is chromatin bound, but no significant enzyme activity could be found in the final nuclear matrix, regardless of the preparation protocol. This result was further substantiated by detailed analysis of histone deacetylases and acetyltransferases during cellular fractionation and nuclear matrix preparation of chicken erythrocytes. Altogether our results suggest that the participation of these enzymes in different nuclear processes may partly be regulated by a distinct location to intranuclear components.

Properties of the yeast nuclear histone deacetylase.

A nuclear histone deacetylase from yeast was partially purified and some of its characteristics were studied. Histone deacetylase activity was stimulated in vitro by high-mobility-group nonhistone chromatin proteins 1 and 2 and ubiquitin and inhibited by spermine and spermidine, whereas n-butyrate had no significant inhibitory effect. Like the mammalian enzyme, partially purified histone deacetylase from yeast was strongly inhibited by trichostatin A. However, in crude extract preparations the yeast enzyme was not inhibited and treatment with trichostatin in vivo did not show any effect, either on the histone acetylation level or on cell viability. At low ionic strength, the enzyme can be isolated as a complex of high molecular mass that is much less inhibited by trichostatin A than is partially purified histone deacetylase activity. Furthermore, radiolabelled oligonucleosomes were more efficiently deacetylated by the complex than by the low-molecular-mass form of the enzyme. The histone deacetylase activity was separated from a polyamine deacetylase activity and its specificity studied. Using h.p.l.c.-purified core histone species as substrate, histone deacetylase from yeast is able to deacetylate all core histones with a slight preference for H3. Our results support the idea that the yeast histone deacetylase may act as a high-molecular-mass complex in vivo.

The role of histones and their modifications in the informative content of chromatin.

It is traditionally accepted that the DNA sequence cannot by itself explain all the mechanisms necessary for the development of living beings, especially in eukaryotes. Indeed part of the information used in these processes is stored in other ways, generally called 'epigenetic', whose molecular mechanisms are mostly unknown. The ultimate explanation for them might reside in the non-DNA moiety of chromatin which may play an active role in heredity ('chromatin information'). Histones are the universal structural component of chromatin. However, recent studies strongly suggest that histones, and their modifications--especially the reversible acetylation of lysines--may act as a recognition signal for regulatory proteins and they may participate, for this reason, in gene regulation. This type of information could be maintained through its replication and, ultimately, it could form the molecular basis of certain processes related to the development of the eukaryotic organisms.

Site specificity of pea histone acetyltransferase B in vitro.

Histone acetyltransferase B from pea embryonic axes has been purified approximately 300-fold by a combination of chromatographic procedures, including affinity chromatography on histone-agarose. The enzyme preparation has been used for the in vitro transfer of acetyl groups from [1-14C]acetyl-CoA to non-acetylated pea histone H4. Up to three acetyl groups can be introduced into the histone. The resulting mono-, di-, and triacetylated H4 isoforms were separated and sequenced to determine the acetylated sites. Only sites 5, 12, and 16 were used by histone acetyltransferase B, but no clear preference among them was observed. The absence of modification of other potentially acetylatable sites is another indication that acetylation of the different lysine residues in the N-terminal H4 tail serves as a specific signal in different nuclear processes.

Histone deacetylase. A key enzyme for the binding of regulatory proteins to chromatin.

Core histones can be modified by reversible, posttranslational acetylation of specific lysine residues within the N-terminal protein domains. The dynamic equilibrium of acetylation is maintained by two enzyme activities, histone acetyltransferase and histone deacetylase. Recent data on histone deacetylases and on anionic motifs in chromatin- or DNA-binding regulatory proteins (e.g. transcription factors, nuclear proto-oncogenes) are summarized and united into a hypothesis which attributes a key function to histone deacetylation for the binding of regulatory proteins to chromatin by a transient, specific local increase of the positive charge in the N-terminal domains of nucleosomal core histones. According to our model, the rapid deacetylation of distinct lysines in especially H2A and H2B would facilitate the association of anionic protein domains of regulatory proteins to specific nucleosomes. Therefore histone deacetylation (histone deacetylases) may represent a unique regulatory mechanism in the early steps of gene activation, in contrast to the more structural role of histone acetylation (histone acetyltransferases) for nucleosomal transitions during the actual transcription process.

Histone acetylation in Zea mays.I. Activities of histone acetyltransferases and histone deacetylases.

DEAE-Sepharose chromatography of extracts from Zea mays meristematic cells revealed multiple histone acetyltransferase and histone deacetylase enzyme forms. An improved method for nuclear isolation allowed us to discriminate nuclear and cytoplasmic enzymes. Two nuclear histone acetyltransferases, A1 and A2, a cytoplasmic B-enzyme and two nuclear histone deacetylases, HD1 and HD2, have been identified. The histone specificity of the different enzyme forms has been studied in an in vitro system, using chicken erythrocyte histones as substrate. The cytoplasmic histone acetyltransferase B is the predominant enzyme, which acetylates mainly histone H4 and to a lesser extent H2A. The nuclear histone acetyltransferase A1 preferentially acetylates H3 and also H4, whereas enzyme A2 is specific for H3. This substrate specificity was confirmed with homologous Z. mays histones. The two histone deacetylases differ from each other with respect to ionic strength dependence, inhibition by acetate and butyrate, and substrate specificity. The strong inhibitory effect of acetate on histone deacetylases was exploited to distinguish different histone acetyltransferase forms.