N-terminal amino acids of bovine alpha interferons are relevant for the neutralization of their antiviral activity

The structure-function relationship of interferons (IFNs) has been studied by epitope mapping. Epitopes of bovine IFNs, however, are practically unknown, despite their importance in virus infections and in the maternal recognition of pregnancy. It has been shown that recombinant bovine (rBo)IFN-alphaC and rBoIFN-alpha1 differ only in 12 amino acids and that the F12 monoclonal antibody (mAb) binds to a linear sequence of residues 10 to 34. We show here that the antiviral activities of these two IFNs were neutralized by the F12 mAb to different extents using two tests. In residual activity tests the antiviral activity dropped by more than 99% with rBoIFN-alphaC and by 84% with rBoIFN-alpha1. In checkerboard antibody titrations, the F12 mAb titer was 12,000 with rBoIFN-alphaC and only 600 with rBoIFN-alpha1. Since these IFNs differ in their amino acid sequence at positions 11, 16 and 19 of the amino terminus, only these amino acids could account for the different neutralization titers, and they should participate in antibody binding. According to the three-dimensional structure described for human and murine IFNs, these amino acids are located in the alpha helix A; amino acids 16 and 19 of the bovine IFNs would be expected to be exposed and could bind to the antibody directly. The amino acid at position 11 forms a hydrogen bond in human IFNs-alpha and it is possible that, in bovine IFNs-alpha, the F12 mAb, binding near position 11, would disturb this hydrogen bond, resulting in the difference in the extent of neutralization observed.

A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide

We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET), expression of a short 6XHis tag at N-terminus (pET3-His) and a high copy number of plasmid (pRSET). The small size of the vector (2.8 kb) and the high copy number/cell (200-250 copies) facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies) and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture). In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site) as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site). Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.

N-terminal-pro-brain natriuretic peptide, but not brain natriuretic peptide, is increased in patients with severe obesity

Elevated body mass index (BMI) has been reported as a risk factor for heart failure. Prevention of heart failure through identification and management of risk factors and preclinical phases of the disease is a priority. Levels of natriuretic peptides as well as activity of their receptors have been found altered in obese persons with some conflicting results. We investigated cardiac involvement in severely obese patients by determining N-terminal-pro-brain natriuretic peptide (NT-proBNP) and brain natriuretic peptide (BNP) and attempting to correlate the levels of these peptides in serum and plasma, respectively, with BMI, duration of obesity, waist circumference, and echocardiographic parameters. Thirty-three patients with severe obesity (mean BMI: 46.39 kg/m², mean age: 39 years) were studied. The control group contained 30 healthy age-matched individuals (BMI: <25 kg/m², mean age: 43 years). The t-test and Spearman correlation were used for statistical analysis. Log-NT-proBNP was significantly higher (P = 0.003) in obese patients (mean 1.67, 95% CI: 1.50-1.83 log pg/mL) compared to controls (mean: 1.32, 95% CI: 1.17-1.47 log pg/mL). The Log-NT-proBNP concentration correlated with duration of obesity (r = 0.339, P < 0.004). No difference was detected in the Log-BNP concentration (P = 0.63) of obese patients (mean: 0.73, 95% CI: 0.46-1.00 log pg/mL) compared to controls (mean: 0.66, 95% CI: 0.51-0.81 log pg/mL). NT-proBNP, but not BNP, is increased in severely obese patients and its concentration in serum is correlated with duration of obesity. NT-proBNP may be useful as an early diagnostic tool for the detection of cardiac burden due to severe obesity.

In silico analysis identifies a C3HC4-RING finger domain of a putative E3 ubiquitin-protein ligase located at the C-terminus of a polyglutamine-containing protein

Almost identical polyglutamine-containing proteins with unknown structures have been found in human, mouse and rat genomes (GenBank AJ277365, AF525300, AY879229). We infer that an identical new gene (RING) finger domain of real interest is located in each C-terminal segment. A three-dimensional (3-D) model was generated by remote homology modeling and the functional implications are discussed. The model consists of 65 residues from terminal position 707 to 772 of the human protein with a total length of 796 residues. The 3-D model predicts a ubiquitin-protein ligase (E3) as a binding site for ubiquitin-conjugating enzyme (E2). Both enzymes are part of the ubiquitin pathway to label unwanted proteins for subsequent enzymatic degradation. The molecular contact specificities are suggested for both the substrate recognition and the residues at the possible E2-binding surface. The predicted structure, of a ubiquitin-protein ligase (E3, enzyme class number 6.3.2.19, CATH code 3.30.40.10.4) may contribute to explain the process of ubiquitination. The 3-D model supports the idea of a C3HC4-RING finger with a partially new pattern. The putative E2-binding site is formed by a shallow hydrophobic groove on the surface adjacent to the helix and one zinc finger (L722, C739, P740, P741, R744). Solvent-exposed hydrophobic amino acids lie around both zinc fingers (I717, L722, F738, or P765, L766, V767, V733, P734). The 3-D structure was deposited in the protein databank theoretical model repository (2B9G, RCSB Protein Data Bank, NJ).

N-terminal prohormone brain natriuretic peptide (NT-proBNP) as a noninvasive marker for restrictive syndromes

Constrictive pericarditis (CP) and restrictive cardiomyopathy share many similarities in both their clinical and hemodynamic characteristics and N-terminal prohormone brain natriuretic peptide (NT-proBNP) is a sensitive marker of cardiac diastolic dysfunction. The objectives of the present study were to determine whether serum NT-proBNP was high in patients with endomyocardial fibrosis (EMF) and CP, and to investigate how this relates to diastolic dysfunction. Thirty-three patients were divided into two groups: CP (16 patients) and EMF (17 patients). The control group consisted of 30 healthy individuals. Patients were evaluated by bidimensional echocardiography, with restriction syndrome evaluated by pulsed Doppler of the mitral flow and serum NT-proBNP measured by immunoassay and detected by electrochemiluminescence. Spearman correlation coefficient was used to analyze the association between log NT-proBNP and echocardiographic parameters. Log NT-proBNP was significantly higher (P < 0.05) in CP patients (log mean: 2.67 pg/mL; 95%CI: 2.43-2.92 log pg/mL) and in EMF patients (log mean: 2.91 pg/mL; 95%CI: 2.70-3.12 log pg/mL) compared with the control group (log mean: 1.45; 95%CI: 1.32-1.60 log pg/mL). There were no statistical differences between EMF and CP patients (P = 0.689) in terms of NT-proBNP. The NT-proBNP log tended to correlate with peak velocity of the E wave (r = 0.439; P = 0.060, but not with A wave (r = -0.399; P = 0.112). Serum NT-proBNP concentration can be used as a marker to detect the presence of diastolic dysfunction in patients with restrictive syndrome; however, serum NT-proBNP levels cannot be used to differentiate restrictive cardiomyopathy from CP.

The N-terminal 33 amino acid domain of Siva-1 is sufficient for nuclear localization

Siva-1 induces apoptosis in multiple pathological processes and plays an important role in the suppression of tumor metastasis, protein degradation, and other functions. Although many studies have demonstrated that Siva-1 functions in the cytoplasm, a few have found that Siva-1 can relocate to the nucleus. In this study, we found that the first 33 amino acid residues of Siva-1 are required for its nuclear localization. Further study demonstrated that the green fluorescent protein can be imported into the nucleus after fusion with these 33 amino acid residues. Other Siva-1 regions and domains showed less effect on Siva-1 nuclear localization. By site-mutagenesis of all of these 33 amino acid residues, we found that mutants of the first 1-18 amino acids affected Siva-1 nuclear compartmentalization but could not complete this localization independently. In summary, we demonstrated that the N-terminal 33 amino acid residues were sufficient for Siva-1 nuclear localization, but the mechanism of this translocation needs additional investigation.

Desarrollo de un método analítico por cromatografía de líquidos de alta resolución para la determinación de ácido fólico en cereales enriquecidos

Tesis (Maestría en Ciencias con Orientación Terminal en Química Biomédica) UANL

Desarrollo y evaluación de un procedimiento de microextracción en fase sólida para la determinación de hidrocarburos aromáticos policíclicos en agua potable

Tesis (Maestría en Ciencias con Orientación Terminal en Química Biomédica) UANL

Rastreo de la actividad antifúngica de plantas del noreste de México, contra los principales agentes causales de micosis pulmonar en la región

Tesis (Maestría en Ciencias con Orientación Terminal en Química Biomédica) UANL.

Implementación de un método de extracción para la determinación de dioxinas en carnes

Tesis (Maestría en Ciencias con Orientación Terminal en Química Biomédica) U.A.N.L. Facultad de Medicina.

Desarrollo y validación de un método de extracción en fase sólida para la determinación de plaguicidas catiónicos (cuats) en alimentos

Tesis (Maestría en Ciencias con Orientación Terminal en Química Biomédica) U.A.N.L., 2006.

Análisis farmacogenético y funcional del gen Timidilato sintasa en cáncer gastrointestinal

Tesis (Maestría en Ciencias con Orientación Terminal en Biología Molecular e Ingeniería Genética) U.A.N.L., 2007.

Análisis del estatus del genoma viral en mujeres infectadas con el virus del papiloma humano.

Tesis (Maestría en Ciencias con Orientación Terminal en Biología Molecular e Ingeniería Genética) UANL, 2011.

Evaluación de la actividad antimicrobiana y antioxidante de aceites esenciales de plantas usadas en medicina tradicional.

Tesis (Maestría en Ciencias con Orientación Terminal en Química Biomédica) UANL, 2011.