Characterization of a catalase-peroxidase variant (L333V-KatG) identified in an INH-resistant Mycobacterium tuberculosis clinical isolate.

Mycobacterium tuberculosis catalase-peroxidase (Mt-KatG) is a bifunctional heme-dependent enzyme that has been shown to activate isoniazid (INH), the widely used antibiotic against tuberculosis (TB). The L333V-KatG variant has been associated with INH resistance in clinical M. tuberculosis isolates from Mexico. To understand better the mechanisms of INH activation, its catalytic properties (catalase, peroxidase, and IN-NAD formation) and crystal structure were compared with those of the wild-type enzyme (WT-KatG). The rate of IN-NAD formation mediated by WT-KatG was 23% greater than L333V-KatG when INH concentration is varied. In contrast to WT-KatG, the crystal structure of the L333V-KatG variant has a perhydroxy modification of the indole nitrogen of W107 from MYW adduct. L333V-KatG shows most of the active site residues in a similar position to WT-KatG; only R418 is in the R-conformation instead of the double R and Y conformation present in WT-KatG. L333V-KatG shows a small displacement respect to WT-KatG in the helix from R385 to L404 towards the mutation site, an increase in length of the coordination bond between H270 and heme Fe, and a longer H-bond between proximal D381 and W321, compared to WT-KatG; these small displacements could explain the altered redox potential of the heme, and result in a less active and stable enzyme.

Bioactive Compounds and Sensory Analysis of Freeze-Dried Prickly Pear Fruits from an Inter-Andean Valley in Peru.

Prickly pear fruits are seasonal and have shades ranging from pale green to deep purple. Their pigments are associated with bioactive compounds, being sensitive to thermal transformation processes for their conservation. The objective of this research was to evaluate the bioactive compounds and the sensory analysis of freeze-dried prickly pear fruits from an inter-Andean valley in Peru. The prickly pear fruits of the morada, anaranjada, and blanca ecotypes came from an inter-Andean valley in Peru at 2972 m altitude. The sliced fruits were freeze-dried at -40 °C and 0.25 mTorr, and the total polyphenol content (TPC), vitamin C, and antioxidant activity (AA) were determined, as well as the color L* a* b*, color index (CI*), FTIR spectra, and mineral content. In the same way, sensory analysis of preferences with nine scales was applied. It was observed that in the freeze-dried fruits, TPC, AA, and vitamin C increased significantly (p-value < 0.05), and their corresponding functional groups increased in intensity in their corresponding FTIR spectra; furthermore, trace elements such as Cu, Fe, Se, Zn, Si, and Mn were identified. On the other hand, freeze-drying provided deeper colors to the fruits, which most panelists said they "very much liked" during the sensory analysis, although the texture was not very well accepted, with most panelists reporting being "indifferent" towards it. The freeze-drying technique allows the bioactive and sensory attributes of prickly pear fruits from inter-Andean valleys to be preserved, making it a potential fruit for export and conservation due to its seasonality.

Engineering a calcium-dependent conformational change in Calbindin D9k by secondary elements replacement.

EF-hand is a common motif in Ca2+-binding proteins, some of which present a conformational change upon Ca2+-binding, a relevant property for signal transduction. In the present work, we investigated the behavior of Calbindin D9k, a modulator protein with a high affinity for Ca2+ but structurally insensitive to its presence. Its non-canoncal N-terminal EF-hand was replaced by chimeric motifs, containing increasing structural elements from the sensor troponin C SCIII motif. We demonstrated that the loop and helix II were the necessary elements for a conformational change promoted by calcium in chimeric Calbindin D9k. Fusion of the isolated chimeric motifs to an activity reporter gene showed the loop as the minimal element to promote a conformational change. The discrepancy between these results is discussed in the light of inter-motif interactions and helix I participation in modulating the Ca2+ affinity and restricting motif conformation.

Engineering Bacillus thuringiensis Cyt1Aa toxin specificity from dipteran to lepidopteran toxicity.

The Cyt and Cry toxins are different pore-forming proteins produced by Bacillus thuringiensis bacteria, and used in insect-pests control. Cry-toxins have a complex mechanism involving interaction with several proteins in the insect gut such as aminopeptidase N (APN), alkaline phosphatase (ALP) and cadherin (CAD). It was shown that the loop regions of domain II of Cry toxins participate in receptor binding. Cyt-toxins are dipteran specific and interact with membrane lipids. We show that Cry1Ab domain II loop3 is involved in binding to APN, ALP and CAD receptors since point mutation Cry1Ab-G439D affected binding to these proteins. We hypothesized that construction of Cyt1A-hybrid proteins providing a binding site that recognizes gut proteins in lepidopteran larvae could result in improved Cyt1Aa toxin toward lepidopteran larvae. We constructed hybrid Cyt1Aa-loop3 proteins with increased binding interaction to Manduca sexta receptors and increased toxicity against two Lepidopteran pests, M. sexta and Plutella xylostella. The hybrid Cyt1Aa-loop3 proteins were severely affected in mosquitocidal activity and showed partial hemolytic activity but retained their capacity to synergize Cry11Aa toxicity against mosquitos. Our data show that insect specificity of Cyt1Aa toxin can be modified by introduction of loop regions from another non-related toxin with different insect specificity.

A reporter system that discriminates EF-hand-sensor motifs from signal-modulators at the single-motif level.

The T-protein is a single-polypeptide bi-functional enzyme composed of a chorismate mutase domain fused to a prephenate dehydrogenase domain (TyrA). We replaced the chorismate mutase domain with canonical or pseudo-Ca(2+)-binding motifs (EF-hand). Canonical-EF-hand-motifs differentiate from pseudo-EF-hand-motifs by experimenting a Ca(2+)-dependent conformational change. The Ca(2+)-free EF-hand-TyrA fusion-proteins showed TyrA activity at the T-protein level. Canonical-EF-hand-TyrA fusions showed a Ca(2+)-dependent loss of TyrA activity, but a pseudo-EF-hand-TyrA fusion showed high TyrA activity level in excess-Ca(2+) conditions. Because TyrA activity exhibits robust changes in response to Ca(2+)-dependent-EF-hand conformational alterations, TyrA could be a good Ca(2+)-reporter enzyme. A chimeric canonical/pseudo-EF-hand strategy is proposed to confer pseudo-EF-hand motifs with a Ca(2+)-dependent conformational change.

Evolution of a new function in an esterase: simple amino acid substitutions enable the activity present in the larger paralog, BioH.

Gene duplication and divergence are essential processes for the evolution of new activities. Divergence may be gradual, involving simple amino acid residue substitutions, or drastic, such that larger structural elements are inserted, deleted or rearranged. Vast protein sequence comparisons, supported by some experimental evidence, argue that large structural modifications have been necessary for certain catalytic activities to evolve. However, it is not clear whether these activities could not have been attained by gradual changes. Interestingly, catalytic promiscuity could play a fundamental evolutionary role: a preexistent secondary activity could be increased by simple amino acid residue substitutions that do not affect the enzyme's primary activity. The promiscuous profile of the enzyme may be modified gradually by genetic drift, making a pool of potentially useful activities that can be selected before duplication. In this work, we used random mutagenesis and in vivo selection to evolve the Pseudomonas aeruginosa PAO1 carboxylesterase PA3859, a small protein, to attain the function of BioH, a much larger paralog involved in biotin biosynthesis. BioH was chosen as a target activity because it provides a highly sensitive selection for evolved enzymatic activities by auxotrophy complementation. After only two cycles of directed evolution, mutants with the ability to efficiently complement biotin auxotrophy were selected. The in vivo and in vitro characterization showed that the activity of one of our mutant proteins was similar to that of the wild-type BioH enzyme. Our results demonstrate that it is possible to evolve enzymatic activities present in larger proteins by discrete amino acid substitutions.

The ß1 domain of protein G can replace the chorismate mutase domain of the T-protein.

T-protein is composed of chorismate mutase (AroQ(T)) fused to the N-terminus of prephenate dehydrogenase (TyrA). Here, we report the replacement of AroQ(T) with the ß1-domain of protein G (Gß1). The TyrA domain shows a strong dehydrogenase activity within the context of this fusion, and our data indicate that Gß1-TyrA folds into a dimeric conformation. Amino acid substitutions in the Gß1 domain of Gß1-TyrA identified residues involved in stabilizing the TyrA dimeric conformation. Gß1 substitutions in the N-terminal ß-hairpin eliminated Gß1-TyrA expression, whereas Gß1-TyrA tolerated Gß1 substitutions in the C-terminal ß-hairpin and in the α-helix. All of the characterized variants folded into a dimeric conformation. The importance of the ß2-strand in forming a Gß1 homo-dimerization interface explains the relevance of the first-ß-hairpin in stabilizing the dimeric TyrA protein.

Sensitive genome-wide screen for low secondary enzymatic activities: the YjbQ family shows thiamin phosphate synthase activity.

Contemporary enzymes are highly efficient and selective catalysts. However, due to the intrinsically very reactive nature of active sites, gratuitous secondary reactions are practically unavoidable. Consequently, even the smallest cell, with its limited enzymatic repertoire, has the potential to carry out numerous additional, very likely inefficient, secondary reactions. If selectively advantageous, secondary reactions could be the basis for the evolution of new fully functional enzymes. Here, we investigated if Escherichia coli has cryptic enzymatic activities related to thiamin biosynthesis. We selected this pathway because this vitamin is essential, but the cell's requirements are very small. Therefore, enzymes with very low activity could complement the auxotrophy of strains deleted of some bona fide thiamin biosynthetic genes. By overexpressing the E. coli's protein repertoire, we selected yjbQ, a gene that complemented a strain deleted of the thiamin phosphate synthase (TPS)-coding gene thiE. In vitro studies confirmed TPS activity, and by directed evolution experiments, this activity was enhanced. Structurally oriented mutagenesis allowed us to identify the putative active site. Remote orthologs of YjbQ from Thermotoga, Sulfolobus, and Pyrococcus were cloned and also showed thiamin auxotrophy complementation, indicating that the cryptic TPS activity is a property of this protein family. Interestingly, the thiE- and yjbQ-coded TPSs are analog enzymes with no structural similarity, reflecting distinct evolutionary origin. These results support the hypothesis that the enzymatic repertoire of a cell such as E. coli has the potential to perform vast amounts of alternative reactions, which could be exploited to evolve novel or more efficient catalysts.

El ascenso del pueblo al poder

Este es un libro propositivo y al mismo tiempo crítico que ciertamente muestra, desde su muy respetable punto de vista, la crisis de legitimidad y gobernabilidad emergente de la dependencia del poder neoliberal en Bolivia. Plantea medidas concretas que permitirían avanzar en democracia e inclusión considerando especialmente que el pueblo ha ascendido legítimamente al Poder. En sus planteamientos subyace que el cambio ya se ha producido y que para que no se frustre, lo que corresponde es administrarlo correcta y eficientemente.

A modified consensus approach to mutagenesis inverts the cofactor specificity of Bacillus stearothermophilus lactate dehydrogenase.

Lactate dehydrogenase from Bacillus stearothermophilus is specific for NAD+. There have been several attempts to alter the cofactor specificity of this enzyme, but these have yielded enzymes with relatively low activities that still largely prefer NAD+. A modified consensus approach was used to create a library of phylogenetically preferred amino acids situated near the cofactor binding site, and variants were screened for their ability to utilize NMN+. A triple mutant (Mut31) was discovered that proved to be more catalytically efficient than wild-type. Mut31 was also better at utilizing NAD+ than the wild-type enzyme and was weakly active with NADP+ and NMN+. An analysis of single amino acid substitutions suggested that all three mutations worked in a concerted fashion to yield robust cofactor utilization. When two previously identified amino acid substitutions were introduced into the Mut31 background, the resultant quintuply substituted enzyme not only utilized NADP+ far better than the wild-type enzyme, it actually inverted its preference for NAD+ and NADP+.

[Frequency and some mortality risk factors in the state of Hidalgo, Mexico, due to neural tube closure defects]. / Frecuencia y algunos factores de riesgo de mortalidad en el estado de Hidalgo, México, por defectos de cierre del tubo neural.

OBJECTIVE: To calculate the risk of fetal death due to neural tube defects and estimate associated factors in the state of Hidalgo, Mexico. MATERIAL AND METHODS: Data were abstracted from death certificates registered during 1990-1995 in the state of Hidalgo, Mexico. The design was a proportional mortality study, which is considered as a variant of the case control design. Cases were deaths with any type of neural tube defect, and controls were fetal deaths due to other causes. RESULTS: A total of 3,673 fetal death certificates were analyzed; 8.06% had neural tube defects and the remaining died of other causes. Fetal death was associated with fetal weight less than 2500 grams (OR 5.0, 95% CI 3.6-6.8), being female (OR 1.7, 95% CI 1.3-2.3), and death during the late fetal period (OR 5.5, 95% CI 3.8-8.1). CONCLUSIONS: Results show that the risk of fetal death due to neural tube defects is greater among low birth weight babies, females, and during the late fetal period.

Frecuencia y algunos factores de riesgo de mortalidad en el estado de Hidalgo, México, por defectos de cierre del tubo neural / Frequency and some mortality risk factors in the state of Hidalgo, Mexico, due to neural tube closure defects

OBJECTIVE: To calculate the risk of fetal death due to neural tube defects and estimate associated factors in the state of Hidalgo, Mexico. MATERIAL AND METHODS: Data were abstracted from death certificates registered during 1990-1995 in the state of Hidalgo, Mexico. The design was a proportional mortality study, which is considered as a variant of the case control design. Cases were deaths with any type of neural tube defect, and controls were fetal deaths due to other causes. RESULTS: A total of 3,673 fetal death certificates were analyzed; 8.06 had neural tube defects and the remaining died of other causes. Fetal death was associated with fetal weight less than 2500 grams (OR 5.0, 95 CI 3.6-6.8), being female (OR 1.7, 95 CI 1.3-2.3), and death during the late fetal period (OR 5.5, 95 CI 3.8-8.1). CONCLUSIONS: Results show that the risk of fetal death due to neural tube defects is greater among low birth weight babies, females, and during the late fetal period.

Increasing the thermal stability of an oligomeric protein, beta-glucuronidase.

The reporter enzyme beta-glucuronidase was mutagenized and evolved for thermostability. After four cycles of screening the best variant was more active than the wild-type enzyme, and retained function at 70 degrees C, whereas the wild-type enzyme lost function at 65 degrees C. Variants derived from sequential mutagenesis were shuffled together, and re-screened for thermostability. The best variants retained activities at even higher temperatures (80 degrees C), but had specific activities that were now less than that of the wild-type enzyme. The mutations clustered near the tetramer interface of the enzyme, and many of the evolved variants showed much greater resistance to quaternary structure disruption at high temperatures, which is also a characteristic of naturally thermostable enzymes. Together, these results suggest a pathway for the evolution of thermostability in which enzymes initially become stable at high temperatures without loss of activity at low temperatures, while further evolution leads to enzymes that have kinetic parameters that are optimized for high temperatures.

Ptosis: clasificación cirugía y resultados / Ptosis: clasification, surgery and results

Se presentan 306 casos operados de ptosis en este Instituto entre 1975 a 1990. De los 176 casos de ptosis mínimas, 167 fueron corregidas con la OFS 1, 4 con OFS 1 modificada por Lauring, y 5 casos con la Quickert. De los 73 casos de ptosis moderada, en 64 se practicó la OFS 2 resecando un tercio del tarso y colocando la sutura alta; 3 casos con Marcus-Gunn asociado fueron corregidos con una OFS 2 modificada, y en 6 pacientes se utilizó la vía cutánea. En las parálisis del tercer par la ptosis severas se trató con suspensión y el procedimiento de Peter. Las grandes resecciones de elevador popr vía cutánea, no dieron buenos resultados y en los últimos casos se practicó de rutina la resección moderada a través de una OFS sin suturas, con éxito notable. Se concluye que aunque no es la OFS una panacea, permite con sus 3 modificaciones, corregir exitosamente casi todos los casos de ptosis adquiridas y congénitas