Geobacillus stearothermophilus V ubiE gene product is involved in the evolution of dimethyl telluride in Escherichia coli K-12 cultures amended with potassium tellurate but not with potassium tellurite

A 3.8-kb fragment of chromosomal DNA of Geobacillus stearothermophilus V cloned in pSP72 (p1VH) confers resistance to potassium tellurite (K₂TeO₃) and to potassium tellurate (K₂TeO₄) when the encoded genes are expressed in Escherichia coli K-12. The nt sequence of the cloned fragment predicts three ORFs of 780, 399, and 600 bp, whose encoded protein products exhibit about 80% similarity with the SUMT methyltransferase and the BtuR protein of Bacillus megaterium, and with the UbiE methyltransferase of Bacillus anthracis A2012, respectively. In addition, E. coli/p1VH cells evolved dimethyl telluride, which was released into the headspace gas above liquid cultures when amended with K₂TeO₃ or with K₂TeO₄. After 48 h of growth in the presence of these compounds, a protein of about 25 kDa was found at a significantly higher level when crude extracts were analyzed by SDS-PAGE. The N-terminal amino acid (aa) sequence of this protein, obtained by Edman degradation, matched the deduced aa sequence predicted by the G. stearothermophilus V ubiE gene. This gene was amplified by PCR, subcloned in pET21b, and transformed into E. coli JM109(DE3). Interestingly, DMTe evolution occurred when these modified cells were grown in K₂TeO₄ – but not in K₂TeO₃ – amended media. These results may be indicative that the two Te oxyanions could be detoxified in the cell by different metabolic pathways.     

Ceruloplasmin, an Indicator of Copper Status

For clinical purposes, the non-ceruloplasmin copper fraction is routinely derived on the basis that ceruloplasmin binds six Cu atoms. However, this approach is limited because the actual ceruloplasmin copper binding is unclear. We performed direct measurement of the total serum copper and ceruloplasmin in 790 healthy individuals. We used an immunoprecipitation technique to separate ceruloplasmin and determined Cu content. With these values, we calculated the Cu/ceruloplasmin (Cp) ratio and thus generated data to support or discard the theoretical calculation of the non-ceruloplasmin fraction. Average of serum Cu and Cp levels were 18.4 +/- 4.4 micromol/l and 390 +/- 100 mg/l, respectively. The immunoprecipitation procedure allowed us to calculate a Cu/Cp ratio of 5.8, respectively, which supports the methodology of calculation that assigns a mean of six copper atoms to each ceruloplasmin molecule. With these values, we calculated that, in apparently normal adults, the non-ceruloplasmin copper (NCPC) fraction is lower than 1.3 micromol/l of Cu. In this report, we examine the Cp/Cu ratio by using Cp immunoprecipitation procedure. Our in vitro and in vivo studies indicate that, as a mean, there are 5.8 atoms of Cu per Cp molecule and that <1.3 micromol/l of Cu would correspond to the NCPC.     

Determination of the taste threshold of copper in water

Copper effects on human health represent a relevant issue in modern nutrition. One of the difficulties in assessing the early, acute effects of copper ingested via drinking water is that the taste of copper may influence the response and the capacity to taste copper in different waters is unknown. The purpose of the study was to determine the taste threshold of copper in different types of water, using soluble and insoluble salts (copper sulfate and copper chloride). Copper-containing solutions (range 1.0-8.0 mg/l Cu) were prepared in tap water, distilled deionized water and uncarbonated mineral water. Sixty-one healthy volunteers (17-50 years of age), with no previous training for sensory evaluation, participated in the study. A modified triangle test was used to define the taste threshold value. The threshold was defined as the lowest copper concentration detected by 50% of the subjects assessed. To evaluate the olfactory input in the threshold value obtained, 15 of 61 subjects underwent a second set of triangle tests with the nose open and clamped, using distilled water with copper sulfate at a concentration corresponding to the individual's threshold. The taste threshold in tap water was 2.6 mg/l Cu for both copper sulfate and copper chloride. The corresponding values for distilled deionized water were 2.4 and 2.5 mg/l Cu for copper sulfate and copper chloride, respectively. In uncarbonated mineral water the threshold values were slightly higher, 3.5 and 3.8 mg/l Cu for copper sulfate and for copper chloride, respectively, which are significantly higher than those observed in tap and distilled waters (P < 0.01, Kruskal-Wallis test). The taste threshold did not change significantly when the nose was clamped. In conclusion, the median values for copper taste threshold were low, ranging between 2.4 and 3.8 mg/l Cu, depending on the type of water.     

Isolation and characterisation of genomic and cDNA clones coding for a serine-, alanine-, and proline-rich protein of Trypanosoma cruzi

We report here the isolation and characterisation of genomic and cDNA clones encoding a Serine-, Alanine-, and Proline-rich protein (SAP) of Trypanosoma cruzi metacyclic trypomastigotes. The deduced peptides translated from these clones were characterised by a high content of residues of alanine, proline, serine, glycine, valine, and threonine distributed in several repeats: P(2-4), S(2-3), A(2-3), AS, SA, PA, AP, SP, PS, and TP. The repeats are partially homologous to the serine-, alanine-, and proline-containing motifs of Leishmania major and Leishmania mexicana proteophosphoglycans. Genes coding for SAP are part of a polymorphic family whose members are linked to members of gp85/sialidase and mucin-like gene families. This is consistent with the hypothesis that this genetic organisation could be a means by which T. cruzi co-ordinates the expression of major surface proteins.     

Ionizing radiation enhances matrix metalloproteinase-2 production in human lung epithelial cells

Radiation pneumonitis is a major complication of radiation therapy. However, the detailed cellular mechanisms have not been clearly defined. Based on the recognition that basement membrane disruption occurs in acute lung injury and that matrix metalloproteinase (MMP)-2 can degrade type IV collagen, one of the major components of the basement membrane, we hypothesized that ionizing radiation would modulate MMP-2 production in human lung epithelial cells. To evaluate this, the modulation of MMP-2 with irradiation was investigated in normal human bronchial epithelial cells as well as in A549 cells. We measured the activity of MMP-2 in the conditioned medium with zymography and the MMP-2 mRNA level with RT-PCR. Both of these cells constitutively expressed 72-kDa gelatinolytic activity, corresponding to MMP-2, and exposure to radiation increased this activity. Consistent with the data of zymography, ionizing radiation increased the level of MMP-2 mRNA. This radiation-induced increase in MMP-2 expression was mediated via p53 because the p53 antisense oligonucleotide abolished the increase in MMP-2 activity as well as the accumulation of p53 after irradiation in A549 cells. These results indicate that MMP-2 expression by human lung epithelial cells is involved in radiation-induced lung injury.     

Diterpenoids from Azorella compacta (Umbelliferae) active on Trypanosoma cruzi

The anti-Trypanosoma cruzi activity of natural products isolated from Azorella compacta was evaluated, with particular emphasis on their effect against intracellular amastigotes. Five diterpenoids from A. compacta derived from mulinane and azorellane were isolated and identified. Only two products, named azorellanol (Y-2) and mulin-11,3-dien-20-oic acid (Y-5), showed trypanocidal activity against all stages of T. cruzi including intracellular amastigotes. At 10 M, these compounds displayed a strong lytic activity. It ranged from 88.4 0.6 to 99.0 1 % for all strains and stages evaluate, with an IC50 /18 h values of 20-84 M and 41-87 M, respectively. The development of intracellular amastigotes was also inhibited by nearly 60% at 25 M. The trypanocidal molecules Y-2 and Y-5 did show different degrees of cytotoxicity depending on the cell line tested, with an IC50 /24 h ranging from 33.2 to 161.2 M. We evaluated the effect of diterpenoids against intracellular T. cruzi forms by immunofluorescent identification of a specific membrane molecular marker (Ssp-4 antigen) of the T. cruzi amastigote forms. The accuracy and reproducibility of the measurements were found to be outstanding when examined by confocal microscopy.     

Evaluation of tumor necrosis factor alpha production in ex vivo short term cultured whole blood from women with polycystic ovary syndrome

In mammals, the pleiotropic biological functions of tumor necrosis factor alpha (TNF-alpha) may include important effects on human reproductive physiology. Thus, chronic anovulation, oligo or amenorrhea, infertility, hyperandrogenism, obesity, insulin resistance and increased TNFalpha serum levels have been observed in women affected by polycystic ovary syndrome (PCOS). Whole blood short - term cell cultures (WBSC) are simple systems where the capacity to produce TNF-alpha by circulating leukocytes, mainly of the macrophage/monocyte lineage, can be accurately quantified. Given the relevance of monocytes/macrophages in the production of TNF-alpha, in this study, in a control-case approach, WBSC from women with PCOS were analyzed in their basal and lipolysaccharide (LPS)- stimulated capacity to produce the cytokine. These measurements did not correlate with the increased serum levels of the cytokine and the normal levels of cortisol, found in PCOS women. Increased serum TNF-alpha levels in PCOS women correlated positively with body mass index and negatively with insulin sensitivity. In spite of the increased serum TNF-alpha levels in PCOS women, basal and LPS stimulated production of the cytokine, by the ex vivo WBSC from these patients, were within normal values.     

RNA splicing in higher plant mitochondria: determination of functional elements in group II intron from a chimeric cox II gene in electroporated wheat mitochondria

Higher plant mitochondria mainly contain group II introns presenting a secondary structure with six helical domains linked to a central hub. Experimental evidence of functional elements in higher plant mitochondria introns is limited since they are unable to undergo self-splicing and the definition of functional domains is based on data obtained from yeast autocatalytic introns. Here we study the role of putative functional elements required for the splicing reaction. The exon-binding and intron-binding sites (EBS and IBS, respectively), and the domain 6, which is involved in lariat formation, were analysed by site-directed mutagenesis and transient expression in electroporated mitochondria. The data presented here demonstrate the role of EBS1-IBS1 and EBS2-IBS2 interactions and reveal a new secondary-structure interaction. The role of the C to U editing conversion in the IBS1 motif is discussed.