Roberto Zúñiga

A dual-specific Glu-tRNAGln and Asp-tRNAAsn amidotransferase is involved in decoding glutamine and asparagine codons in Acidithiobacillus ferrooxidans

The gatC, gatA and gatB genes encoding the three subunits of glutamyl‐tRNAGln amidotransferase from Acidithiobacillus ferrooxidans, an acidophilic bacterium used in bioleaching of minerals, have been cloned and expressed in Escherichia coli. As in Bacillus subtilis the three gat genes are organized in an operon‐like structure in A. ferrooxidans. The heterologously overexpressed enzyme converts Glu‐tRNAGln to Gln‐tRNAGln and Asp‐tRNAAsn to Asn‐tRNAAsn. Biochemical analysis revealed that neither glutaminyl‐tRNA synthetase nor asparaginyl‐tRNA synthetase is present in A. ferrooxidans, but that glutamyl‐tRNA synthetase and aspartyl‐tRNA synthetase enzymes are present in the organism. These data suggest that the transamidation pathway is responsible for the formation of Gln‐tRNA and Asn‐tRNA in A. ferrooxidans.

Interleukin 10 decreases MICA expression on melanoma cell surface.

Natural‐killer group 2, member D (NKG2D) binds to a variety of ligands, including the major histocompatibility complex (MHC) class I chain‐related proteins (MIC) and UL16‐binding proteins (ULBP). It is regarded as a co‐activating receptor on NK cells, having an important role in the cell‐mediated immune response to tumours. We studied the influence of interleukin (IL)‐10 on the regulation of MIC and ULBP expression on melanoma cells, and its effect on the cytotoxic function of NK cells in vitro. Here, we show that, in the presence of IL‐10, FMS mel and BL mel cell lines decreased MICA and ULBP2 surface expression, whereas MHC class I did not change substantially on the cell surface. MICA mRNA levels decreased in IL‐10‐treated FMS and IL‐10‐transduced BL cell lines. Interestingly, we observed that MICB surface expression and its mRNA levels increased upon IL‐10 treatment in a melanoma cell line. These changes in NKG2D ligands surface expression patterns owing to IL‐10 treatment resulted in an effect on lysis susceptibility mediated by lymphocyte‐activated killer cells, as tumour cell lines that displayed a higher decrease of MICA on their surface had lower levels of lysis. In addition, expression of CD107a was downregulated on the surface of NK cells following stimulation with IL‐10‐treated FMS cells. Our results suggest a novel function for IL‐10 in the modulation of NKG2D ligand expression and in the control of cytotoxicity mediated by NKG2D/NKG2D ligand axis.

An efficient method for variable region assembly in the construction of scFv phage display libraries using independent strand amplification

Phage display library technology is a common method to produce human antibodies. In this technique, the immunoglobulin variable regions are displayed in a bacteriophage in a way that each filamentous virus displays the product of a single antibody gene on its surface. From the collection of different phages, it is possible to isolate the virus that recognizes specific targets. The most common form in which to display antibody variable regions in the phage is the single chain variable fragment format (scFv), which requires assembly of the heavy and light immunoglobulin variable regions in a single gene. In this work, we describe a simple and efficient method for the assembly of immunoglobulin heavy and light chain variable regions in a scFv format. This procedure involves a two-step reaction: (1) DNA amplification to produce the single strand form of the heavy or light chain gene required for the fusion; and (2) mixture of both single strand products followed by an assembly reaction to construct a complete scFv gene. Using this method, we produced 6-fold more scFv encoding DNA than the commonly used splicing by overlap extension PCR (SOE-PCR) approach. The scFv gene produced by this method also proved to be efficient in generating a diverse scFv phage display library. From this scFv library, we obtained phages that bound several non-related antigens, including recombinant proteins and rotavirus particles.

Conserved amino acids near the carboxy terminus of bacterial tyrosyl-tRNA synthetase are involved in tRNA and Tyr-AMP binding

Bacterial tyrosyl‐tRNA synthetases occur in two large subfamilies, TyrRS and TyrRZ, that possess about 25% amino acid identity. Their amino‐terminal region, the active site domain, is more conserved (>36% identity). The carboxy‐terminal segment of these enzymes includes the tRNA binding domain and contains only few conserved residues. Replacement of three of these residues in Acidithiobacillus ferrooxidans TyrRZ revealed that S356 and K395 play roles in tRNA binding, while H306, a residue at the junction of the catalytic and tRNA binding domains, stabilizes the Tyr‐AMP:TyrRZ complex. The replacement data suggest that conserved amino acids in A. ferrooxidans TyrRZ and Bacillus stearothermophilus TyrRS play equivalent roles in enzyme function.

Clinical significance of tumor expression of major histocompatibility complex class I-related chains A and B (MICA/B) in gastric cancer patients

Gastric cancer (GC) is the third most common cause of cancer death worldwide. Natural killer cells play an important role in the immune defense against transformed cells. They express the activating receptor NKG2D, whose ligands belong to the MIC and ULBP/RAET family. Although it is well established that these ligands are generally expressed in tumors, the association between their expression in the tumor and gastric mucosa and clinical parameters and prognosis of GC remains to be addressed. In the present study, MICA and MICB expression was analyzed, by flow cytometry, in 23 and 20 pairs of gastric tumor and adjacent non-neoplasic gastric mucosa, respectively. Additionally, ligands expression in 13 tumors and 7 gastric mucosa samples from GC patients were evaluated by immunohistochemistry. The mRNA levels of MICA in 9 pairs of tumor and mucosa were determined by quantitative PCR. Data were associated with the clinicopathological characteristics and the patient outcome. MICA expression was observed in 57% of tumors (13/23) and 44% of mucosal samples (10/23), while MICB was detected in 50% of tumors (10/20) and 45% of mucosal tissues (9/20). At the protein level, ligand expression was significantly higher in the tumor than in the gastric mucosa. MICA mRNA levels were also increased in the tumor as compared to the mucosa. However, clinicopathological analysis indicated that, in patients with tumors >5 cm, the expression of MICA and MICB in the tumor did not differ from that of the mucosa, and tumors >5 cm showed significantly higher MICA and MICB expression than tumors ≤5 cm. Patients presenting tumors >5 cm that expressed MICA and MICB had substantially shorter survival than those with large tumors that did not express these ligands. Our results suggest that locally sustained expression of MICA and MICB in the tumor may contribute to the malignant progression of GC and that expression of these ligands predicts an unfavorable prognosis in GC patients presenting large tumors.

A dispensable peptide from Acidithiobacillus ferrooxidans tryptophanyl-tRNA synthetase affects tRNA binding

The activation domain of class I aminoacyl‐tRNA synthetases, which contains the Rossmann fold and the signature sequences HIGH and KMSKS, is generally split into two halves by the connective peptides (CP1, CP2) whose amino acid sequences are idiosyncratic. CP1 has been shown to participate in the binding of tRNA as well as the editing of the reaction intermediate aminoacyl‐AMP or the aminoacyl‐tRNA. No function has been assigned to CP2. The amino acid sequence of Acidithiobacillus ferrooxidans TrpRS was predicted from the genome sequence. Protein sequence alignments revealed that A. ferrooxidans TrpRS contains a 70 amino acids long CP2 that is not found in any other bacterial TrpRS. However, a CP2 in the same relative position was found in the predicted sequence of several archaeal TrpRSs. A. ferrooxidans TrpRS is functional in vivo in Escherichia coli. A deletion mutant of A. ferrooxidans trpS lacking the coding region of CP2 was constructed. The in vivo activity of the mutant TrpRS in E. coli, as well as the kinetic parameters of the in vitro activation of tryptophan by ATP, were not altered by the deletion. However, the K m value for tRNA was seven‐fold higher upon deletion, reducing the efficiency of aminoacylation. Structural modeling suggests that CP2 binds to the inner corner of the L shape of tRNA.

Valorización Agrícola de Purines Porcinos Procesados con Aserrín de Pino

In order to control excessive humidity of swine manure and to obtain a stabilized material, pine sawdust was used as bulk material. Several doses of pine sawdust were evaluated: 1.0, 1.5 and 2.0 m 3 . Raw manure was spread and maintained in rest for five days. Piles of this material were constructed under aerobic conditions and periodically turned. At the end of the assay all treatments showed a decrease of the salinity. The high contents of organic matter and the C/N ratio of these mixtures, and the decrease of the humidity of the bulk material allows assuming that this material could be reused with new charges of swine manure. The end product obtained has appropriate characteristics in terms of ammonium/nitrate ratio for establishing cultures without restrictions and the fecal coliform levels decrease drastically.

Mild hypothermia upregulates myc and xbp1s expression and improves anti-TNFα production in CHO cells

Chinese hamster ovary (CHO) cells are the most frequently used host for commercial production of therapeutic proteins. However, their low protein productivity in culture is the main hurdle to overcome. Mild hypothermia has been established as an effective strategy to enhance protein specific productivity, although the causes of such improvement still remain unclear. The self-regulation of global transcriptional regulatory factors, such as Myc and XBP1s, seems to be involved in increased the recombinant protein production at low temperature. This study evaluated the impact of low temperature in CHO cell cultures on myc and xbp1s expression and their effects on culture performance and cell metabolism. Two anti-TNFα producing CHO cell lines were selected considering two distinct phenotypes: i.e. maximum cell growth, (CN1) and maximum specific anti-TNFα production (CN2), and cultured at 37, 33 and 31°C in a batch system. Low temperature led to an increase in the cell viability, the expression of the recombinant anti-TNFα and the production of anti-TNFα both in CN1 and CN2. The higher production of anti-TNFα in CN2 was mainly associated with the large expression of anti-TNFα. Under mild hypothermia myc and xbp1s expression levels were directly correlated to the maximal viable cell density and the specific anti-TNFα productivity, respectively. Moreover, cells showed a simultaneous metabolic shift from production to consumption of lactate and from consumption to production of glutamine, which were exacerbated by reducing culture temperature and coincided with the increased anti-TNFα production. Our current results provide new insights of the regulation of myc and xbp1s in CHO cells at low temperature, and suggest that the presence and magnitude of the metabolic shift might be a relevant metabolic marker of productive cell line.

STAT3 inhibition by STA21 increases cell surface expression of MICB and the release of soluble MICB by gastric adenocarcinoma cells

NKG2D is an activating receptor expressed on NK cells that binds to a variety of ligands, including MICA and MICB. These cell surface glycoproteins are overexpressed under cellular transformation, thus playing an important role in cell-mediated immune response to tumors. STAT3 is a transcription factor that is constitutively active in cancer. It negatively regulates MICA expression on target cells, while its inhibition enhances NK cell cytotoxicity against tumors. In this work, we aimed to describe the effect of STAT3 signaling inhibition by STA21 on the regulation of MICB expression in gastric adenocarcinoma cells and its effect on the cytotoxic function of NK cells. Treatment of gastric adenocarcinoma cells with STA21 induced an increase in MICB expression and soluble MICB secretion, as well as a variable pattern on effector cell degranulation. Soluble MICB secretion by gastric adenocarcinoma cells was not affected by metalloprotease inhibition. We also observed that primary gastric adenocarcinoma tissue released soluble MICB into the extracellular milieu. Recombinant MICB induced a significant decrease in the levels of NKG2D receptor on effector NK and CD8+ T cells, which correlated with an impaired cytotoxic function. Altogether, our data provide evidence that STAT3 signaling pathway regulates MICB expression on gastric adenocarcinoma cells and that recombinant soluble MICB compromises the cytolytic activity of NK cells.