Clementina Castro

Structure of a microbial homologue of mammalian platelet-activating factor acetylhydrolases: Streptomyces exfoliatus lipase at 1.9 å resolution

BACKGROUND Neutral lipases are ubiquitous and diverse enzymes. The molecular architecture of the structurally characterized lipases is similar, often despite a lack of detectable homology at the sequence level. Some of the microbial lipases are evolutionarily related to physiologically important mammalian enzymes. For example, limited sequence similarities were recently noted for the Streptomyces exfoliatus lipase (SeL) and two mammalian platelet-activating factor acetylhydrolases (PAF-AHs). The determination of the crystal structure of SeL allowed us to explore the structure-function relationships in this novel family of homologous hydrolases. RESULTS The crystal structure of SeL was determined by multiple isomorphous replacement and refined using data to 1.9 A resolution. The molecule exhibits the canonical tertiary fold of an alpha/beta hydrolase. The putative nucleophilic residue, Ser131, is located within a nucleophilic elbow and is hydrogen bonded to His209, which in turn interacts with Asp177. These three residues create a triad that closely resembles the catalytic triads found in the active sites of other neutral lipases. The mainchain amides of Met132 and Phe63 are perfectly positioned to create an oxyanion hole. Unexpectedly, there are no secondary structure elements that could render the active site inaccessible to solvent, like the lids that are commonly found in neutral lipases. CONCLUSIONS The crystal structure of SeL reinforces the notion that it is a homologue of the mammalian PAF-AHs. We have used the catalytic triad in SeL to model the active site of the PAF-AHs. Our model is consistent with the site-directed mutagenesis studies of plasma PAF-AH, which implicate Ser273, His351 and Asp296 in the active site. Our study therefore provides direct support for the hypothesis that the plasma and isoform II PAF-AHs are triad-containing alpha/beta hydrolases.

Sequence and functional analysis of the Streptomyces phaeochromogenes plasmid pJV1 reveals a modular organization of Streptomyces plasmids that replicate by rolling circle.

pJV1 is an 11 kb, high-copy-number conjugative Streptomyces phaeochromogenes plasmid that replicates by the rolling circle mechanism (RCR). Sequencing combined with functional analysis of deletion, insertion and frameshift mutations was used to characterize the genes involved in plasmid transfer and chromosome mobilization (Cma), the single-strand origin for RCR and an associated strong incompatibility (Sti) determinant. pJV1 contains two essential transfer genes whose expression is regulated by an adjacent repressor gene with similarity to the GntR family of regulators. A consensus sequence specific for the helix-turn-helix motifs of repressor proteins of Streptomyces plasmids is proposed. Unregulated expression of the transfer genes by inactivation of the repressor is lethal. Three additional genes increase intramycelial plasmid spread resulting in pock formation but, unlike the essential transfer genes, are not required for Cma. The pJV1 transfer genes and their regulatory region, but not the minimal replication region encoding the double-strand replication origin and replication protein, are similar in their sequence and arrangement to those of the Streptomyces nigrifaciens plasmid pSN22, revealing a modular organization of Streptomyces RCR plasmids.

Trypanosoma cruzi ribosomal RNA: internal break in the large-molecular-mass species and number of genes.

The large-molecular-mass ribosomal ribonucleic acid from Trypanosoma cruzi probably contains an internal break. The molecule can be obtained in its intact form or in its two fragments depending on the denaturing agents used for its purification and/or display. This break appears to be an in vivo late processing step rather than a random nucleolytic cleavage during in vitro manipulations. Calculations of mass, from gel electrophoretograms, for the large and small main ribosomal ribonucleic acid species and for the two chains derived from the large species gave values of 1.37, 0.84, 0.70 and 0.57 X 10(6) daltons, respectively. Sedimentation velocity measurements in sucrose gradients and in the analytical ultracentrifuge indicated sedimentation coefficients of 24 and 18 S for the large and small main species, respectively. Saturation hybridization curves showed that the nuclear genome, quantified by chemical analysis, contains about 114 ribosomal ribonucleic acid gene copies.

Genome organization and ploidy number in Trypanosoma cruzi.

Trypanosoma cruzi total DNA was analyzed by DNA:DNA reassociation kinetics. The nonlinear least-squares computer solution could reasonably be fitted to three second-order kinetic components. The highly repetitive, middle repetitive, and single copy components comprised 9, 35 and 49% of the genome, respectively. The single copy sequences showed a kinetic complexity of approximately 4 times that of Escherichia coli and of some 11,000 average-sized structural genes. The repetitive sequences (about 6900) presented the long-period pattern of interspersion with a modal length of 7800 bases. The kinetic complexity of total DNA was compatible with a value of at least a diploid genome per cell.

Sequence of the Streptomyces albus G lipase-encoding gene reveals the presence of a prokaryotic lipase family.

An extracellular lipase (Lip)-encoding gene from Streptomyces albus G has been cloned and sequenced. It encodes a Lip with 82% sequence identity to another previously cloned Lip from a Streptomyces species not closely related. These two sequences can be aligned with 33% identity to the sequence of Lip1 from the antarctic psychrotroph Moraxella TA144 [G. Feller et al., Nucleic Acids Res. 18 (1990) 6431]. An alignment of the three sequences revealed amino-acid substitutions which might be responsible for the greater thermal stability of the Streptomyces lipases. The presence of this lip gene family in several members of the Streptomyces genus was also shown.

Significant Clinical Impact of Recurrent BRCA1 and BRCA2 Mutations in Mexico

Frequent recurrent mutations in the breast and ovarian cancer susceptibility (BRCA) genes BRCA1 and BRCA2 among Hispanics, including a large rearrangement Mexican founder mutation (BRCA1 exon 9‐12 deletion [ex9‐12del]), suggest that an ancestry‐informed BRCA‐testing strategy could reduce disparities and promote cancer prevention by enabling economic screening for hereditary breast and ovarian cancer in Mexico.

Association between ERCC1 and XPA expression and polymorphisms and the response to cisplatin in testicular germ cell tumours

Background:Cisplatin cures over 80% of testicular germ cell tumours (TGCTs), and nucleotide-excision repair (NER) modifies the sensitivity to cisplatin. We explored the association between NER proteins and their polymorphisms with cisplatin sensitivity (CPS) and overall survival (OS) of patients with non-seminomatous (ns)-TGCTs.Methods:The expression of ERCC1 and XPA and the presence of γH2AX were evaluated in cancer cell lines and in fresh ns-TGCTs. The ERCC1 protein was also determined in ns-TGCTs. The differences between CPS and non-CPS cell lines and patients were analysed by Student’s t- or χ2-tests. The differences in OS were analysed using the log-rank test, and the hazard ratios (HRs) were calculated using the Cox model.Results:High ERCC1 expression was observed in the non-CPS cells, and both ERCC1 and γH2AX expressions were augmented after cisplatin treatment. Increased ERCC1 expression was also identified in non-CPS patients. Neither polymorphism was associated with either CPS or OS. The presence of ERCC1 was associated with non-CPS (P=0.05) and adjusted in the prognosis groups. The HR in ERCC1-negative and non-CPS patients was >14.43, and in ERCC1-positive and non-CPS patients the HR was >11.86 (P<0.001).Conclusions:High levels of ERCC1 were associated with non-CPS, suggesting that ERCC1 could be used as a potential indicator of the response to cisplatin and prognosis in ns-TGCTs.

Disruption of CTCF at the miR-125b1 locus in gynecological cancers

In cancer cells, transcriptional gene silencing has been associated with genetic and epigenetic defects. The disruption of DNA methylation patterns and covalent histone marks has been associated with cancer development. Until recently, microRNA (miRNA) gene silencing was not well understood. In particular, miR-125b1 has been suggested to be an miRNA with tumor suppressor activity, and it has been shown to be deregulated in various human cancers. In the present study, we evaluated the DNA methylation at the CpG island proximal to the transcription start site of miR-125b1 in cancer cell lines as well as in normal tissues and gynecological tumor samples. In addition, we analyzed the association of CTCF and covalent histone modifications at the miR-125b1 locus. To assess the DNA methylation status of the miR-125b1, genomic DNA was transformed with sodium bisulfite, and then PCR-amplified with modified primers and sequenced. The miR-125b1 gene expression was analyzed by qRT-PCR using U6 as a control for constitutive gene expression. CTCF repressive histone marks abundance was evaluated by chromatin immunoprecipitation assays. The disruption of CTCF in breast cancer cells correlated with the incorporation of repressive histone marks such H3K9me3 and H3K27me3 as well as with aberrant DNA methylation patterns. To determine the effect of DNA methylation at the CpG island of miR-125b1 on the expression of this gene, we performed a qRT-PCR assay. We observed a significant reduction on the expression of miR-125b1 in cancer cells in comparison with controls, suggesting that DNA methylation at the CpG island might reduce miR-125b1 expression. These effects were observed in other gynecological cancers, including ovarian and cervical tumors. A reduction of miR-125b1 expression in cancers, correlated with methylation, repressive histone marks and loss of CTCF binding at the promoter region.BackgroundIn cancer cells, transcriptional gene silencing has been associated with genetic and epigenetic defects. The disruption of DNA methylation patterns and covalent histone marks has been associated with cancer development. Until recently, microRNA (miRNA) gene silencing was not well understood. In particular, miR-125b1 has been suggested to be an miRNA with tumor suppressor activity, and it has been shown to be deregulated in various human cancers. In the present study, we evaluated the DNA methylation at the CpG island proximal to the transcription start site of miR-125b1 in cancer cell lines as well as in normal tissues and gynecological tumor samples. In addition, we analyzed the association of CTCF and covalent histone modifications at the miR-125b1 locus.MethodsTo assess the DNA methylation status of the miR-125b1, genomic DNA was transformed with sodium bisulfite, and then PCR-amplified with modified primers and sequenced. The miR-125b1 gene expression was analyzed by qRT-PCR using U6 as a control for constitutive gene expression. CTCF repressive histone marks abundance was evaluated by chromatin immunoprecipitation assays.ResultsThe disruption of CTCF in breast cancer cells correlated with the incorporation of repressive histone marks such H3K9me3 and H3K27me3 as well as with aberrant DNA methylation patterns. To determine the effect of DNA methylation at the CpG island of miR-125b1 on the expression of this gene, we performed a qRT-PCR assay. We observed a significant reduction on the expression of miR-125b1 in cancer cells in comparison with controls, suggesting that DNA methylation at the CpG island might reduce miR-125b1 expression. These effects were observed in other gynecological cancers, including ovarian and cervical tumors.ConclusionsA reduction of miR-125b1 expression in cancers, correlated with methylation, repressive histone marks and loss of CTCF binding at the promoter region.

Association of the estrogen receptor α gene polymorphisms with osteoporosis in the Mexican population

The estrogen receptor gene (ERα) has been implicated in the development of osteoporosis. In this study, the association of two ERα gene polymorphic markers (a TA dinucleotide repeat and a single nucleotide polymorphism, G2014A) with osteoporosis was tested in 70 osteoporotic women, 70 non‐osteoporotic women and 500 subjects from the Mexican population. According to the genetic analysis of the Mexican population using eight unlinked polymorphic markers, we found that our population is structured into three subpopulations; therefore, the allele–phenotype relationship was analyzed with a statistical method that considered population stratification. We found that the G2014A polymorphism is associated with the presence of osteoporosis while the TA dinucleotide repeat is not. The G allele and the GG genotype frequencies of the G2014A marker were significantly higher in osteoporotic than in non‐osteoporotic women. Likewise, subjects bearing the G allele in heterozygous or homozygous displayed lower values for lumbar bone mineral density and T score than those who did not present any G allele. The effect of confounders for osteoporosis on the association of G allele–osteoporosis was ruled out. In summary, we conclude that the G2014 polymorphism may become a useful marker for genetic studies of osteoporosis in the Mexican population.

Methylenetetrahydrofolate Reductase Polymorphism (677 C>T) Predicts Long Time to Progression in Metastatic Colon Cancer Treated with 5-Fluorouracil and Folinic Acid

BACKGROUND AND AIMS Fluoropyrimidine-based chemotherapy is the most common treatment for unresectable metastatic colorectal cancer (m-CRC). Therapy with 5-FU/folinic acid (FA) continues to be a standard treatment in developing countries. Pharmacogenomics allows the tailoring of cancer therapy to the patient. The polymorphism 677C>T of the methylenetetrahydrofolate reductase (MTHFR) gene seems to influence the effectiveness of treatment with 5-FU. We undertook this study to evaluate the frequency of MTHFR 677C>T polymorphism and its relationship to the time to progression (TTP) and overall survival (OS) in m-CRC treated with 5-FU/FA. METHODS The MTHFR 677C>T polymorphism was determined using PCR and allele-specific digestion. The clinical variables, TTP and OS, were analyzed in each case and compared between wild-type and variant polymorphic groups. RESULTS Among 34 patients (12 males and 22 females), we detected eight wild-type homozygous patients (CC; 24%), nine variant homozygous (TT; 26%), and 17 heterozygous (CT; 50%) individuals. The median TTP in patients with the MTHFR 677 CC, CT, and TT genotypes was 3.43, 4.77, and 4.80 months, respectively (p = 0.047, log rank). A longer TTP was observed in patients with polymorphic variant (CT and TT) compared with the wild-type homozygous patients (4.80 vs. 3.43 months; p = 0.031, log rank). CONCLUSIONS In this study, the frequency of the MTHFR 677C>T polymorphism is 50% among m-CRC Mexican patients. The results of this study appear to show that the presence of the MTHFR 677C>T polymorphism is associated with longer TTP and OS in m-CRC treated with 5-FU/FA.