Biomedical subjects

F Valle

Publications and source records attributed to F Valle.

At least 19 recordsLinked to original sources

PubMed2002

AFM structural study of the molecular chaperone GroEL and its two-dimensional crystals: an ideal "living" calibration sample.

Supramolecular complexes, such as chaperonins, are suitable samples for atomic force microscope structural studies because they have a very well defined shape. High-resolution images can be made using tapping mode in liquid under native conditions. Details about the two-dimensional structures formed onto the surface upon adsorption and of the single protein can be observed. Dissection of the upper ring of the supramolecular complex as a result of the applied lateral force through scanning tip is observed. Finally, the combination of lateral convolution and tip penetration into the cavity of chaperonins offers a direct evaluation of the tip convolution effect on images of macromolecular samples.

Calibration↗

PubMed2001

Single-molecule imaging by atomic force microscopy of the native chaperonin complex of the thermophilic archaeon Sulfolobus solfataricus.

The chaperonin of the extremely thermophilic archaeon Sulfolobus solfataricus has been imaged for the first time under native conditions using the atomic force microscope. This technique allows to visualize the structure of biomolecules in solution under physiological conditions providing a nanometer resolution topographic image of the sample. Single molecule studies can reveal fine structural details, providing a powerful insight into the active conformation of a macromolecule, and also allowing to detect different conformational states corresponding to functional changes.

Archaeal Proteins↗

PubMed2001

Construction of protein overproducer strains in Bacillus subtilis by an integrative approach.

We evaluated the effect of several genetic factors reported as having a role in the induction of the expression of significant levels of recombinant protein in Bacillus subtilis. We utilized the beta-galactosidase reporter protein from Escherichia coli as our model for measuring the overproduction of heterologous proteins in B. subtilis. The lacZ gene was expressed in B. subtilis using the regulatory region of the subtilisin gene aprE. In this study, we considered factors known to modulate the transcription and translation initiation rates and genetic and mRNA stability. We also consider the effects of different genetic backgrounds, such as degU32 and hpr2, that until now have been studied independently. By changing the native -35 promoter box to the consensus TTGACA sequence of the aprE promoter, a significant 100-fold increase in the beta-galactosidase activity was obtained. On the other hand, changes such as the GTG to ATG start codon, the construction of a consensus AAGGAGG ribosome binding site, and the addition of the cryIIIA transcription terminator at the 3' end of the lacZ gene, produced only marginal effects on the final beta-galactosidase activity.

Bacillus subtilis↗

PubMed2001

Metabolic profiles and aprE expression in anaerobic cultures of Bacillus subtilis using nitrate as terminal electron acceptor.

Cultures using nitrate as the terminal electron acceptor were conducted in Schaeffer's medium to evaluate the growth performance and metabolic profiles of Bacillus subtilis, and its potential to express the aprE (subtilisin) gene under anoxic conditions. Nitrate was converted to ammonia through nitrite reduction; and different product profiles were observed during the growth phase when nitrate was added at various concentrations (4-24 mM) to Schaeffer's medium containing glucose (4 g l(-1)). If nitrate was not limiting, then acetic acid and acetoin were accumulated, suggesting a limitation of reduced cofactors but, if nitrate became limiting, then lactic acid and butanediol were accumulated, suggesting an excess of reduced cofactors. Due to a strong lysis at the onset of the end of the growth phase, sporulation frequency and aprE expression were negligible in anaerobic batch cultures. Fed-batch fermentation allowed the development of a stationary phase through a continuous supply of glucose and nitrate. In this case, sporulation frequency was almost null, but interestingly aprE expression was similar to that found in aerobic cultures.

Acetic Acid↗

PubMed2001

Characterization of sugar mixtures utilization by an Escherichia coli mutant devoid of the phosphotransferase system.

Due to catabolite repression in microorganisms, sugar mixtures cannot be metabolized in a rapid and efficient manner. Therefore, the development of mutant strains that avoid this regulatory system is of special interest to fermentation processes. In the present study, the utilization of sugar mixtures by an Escherichia coli mutant strain devoid of the phosphotransferase system (PTS) was characterized. This mutant can transport glucose (PTS- Glucose+ phenotype) by a non-PTS mechanism as rapidly as its wild-type parental strain. In cultures grown in minimal medium supplemented with glucose-xylose or glucose-arabinose mixtures, glucose repressed arabinose- or xylose-utilization in the wild-type strain. However, under the same culture conditions with the PTS- Glucose+ mutant, glucose and arabinose were co-metabolized, but glucose still exerted a partial repressive effect on xylose consumption. In cultures growing with a triple mixture of glucose-arabinose-xylose, the wild-type strain sequentially utilized glucose, arabinose and finally, xylose. In contrast, the PTS- Glucose+ strain co-metabolized glucose and arabinose, whereas xylose was utilized after glucose-arabinose depletion. As a result of glucose-arabinose co-metabolism, the PTS- Glucose+ strain consumed the total amount of sugars contained in the culture medium 16% faster than the wild-type strain. [14C]-Xylose uptake experiments showed that in the PTS- Glucose+ strain, galactose permease increases xylose transport capacity and the observed partial repression of xylose utilization depends on the presence of intracellular glucose.

Carbohydrate Metabolism↗

PubMed2001

Imaging the native structure of the chaperone protein GroEL without fixation using atomic force microscopy.

Most sample preparation methods for scanning probe or electron microscopy require that biomolecules, such as proteins, be fixed. Fixation destroys the molecular functionality and can possibly affect the true molecular structure. Here we report sample preparation conditions that allow the imaging of an unfixed protein, GroEL, under in-vivo conditions, by atomic force microscopy. Under these conditions, the protein should maintain its native structure and biological activity. The typical toroidal shape with pore of the GroEL complex was easily visible in the images. Images of a single complex show dimensions that agree well with crystallographic data. Under in-vivo conditions, it should be possible to study the biological activity and function of proteins.

Chaperonin 60↗

PubMed2001

Rapid evolution of novel traits in microorganisms.

The use of natural microorganisms in biotransformations is frequently constrained by their limited tolerance to the high concentrations of metabolites and solvents required for effective industrial production. In many cases, more robust strains have to be generated by random mutagenesis and selection. This process of directed evolution can be accelerated in mutator strains, which carry defects in one or more of their DNA repair genes. However, in order to use mutator strains, it is essential to restore the normal low mutation rate of the selected organisms immediately after selection to prevent the accumulation of undesirable spontaneous mutations. To enable this process, we constructed temperature-sensitive plasmids that temporarily increase the mutation frequency of their hosts by 20- to 4,000-fold. Under appropriate selection pressure, microorganisms transformed with mutator plasmids can be quickly evolved to exhibit new, complex traits. By using this approach, we were able to increase the tolerance of three bacterial strains to dimethylformamide by 10 to 20 g/liter during only two subsequent transfers. Subsequently, the evolved strains were returned to their normal low mutation rate by curing the cells of the mutator plasmids. Our results demonstrate a new and efficient method for rapid strain improvement based on in vivo mutagenesis.

Dimethylformamide↗

PubMed2000

Functional interaction of proliferating cell nuclear antigen with MSH2-MSH6 and MSH2-MSH3 complexes.

Eukaryotic DNA mismatch repair requires the concerted action of several proteins, including proliferating cell nuclear antigen (PCNA) and heterodimers of MSH2 complexed with either MSH3 or MSH6. Here we report that MSH3 and MSH6, but not MSH2, contain N-terminal sequence motifs characteristic of proteins that bind to PCNA. MSH3 and MSH6 peptides containing these motifs bound PCNA, as did the intact Msh2-Msh6 complex. This binding was strongly reduced when alanine was substituted for conserved residues in the motif. Yeast strains containing alanine substitutions in the PCNA binding motif of Msh6 or Msh3 had elevated mutation rates, indicating that these interactions are important for genome stability. When human MSH3 or MSH6 peptides containing the PCNA binding motif were added to a human cell extract, mismatch repair activity was inhibited at a step preceding DNA resynthesis. Thus, MSH3 and MSH6 interactions with PCNA may facilitate early steps in DNA mismatch repair and may also be important for other roles of these eukaryotic MutS homologs.

Amino Acid Sequence↗

PubMed2000

A family of removable cassettes designed to obtain antibiotic-resistance-free genomic modifications of Escherichia coli and other bacteria.

Modifications of microbial genomes often require the use of the antibiotic-resistance (Anb(R))-encoding genes and other easily selectable markers. We have developed a set of such selectable markers (Cm(R), Km(R) and Gm(R)), which could easily be inserted into the genome and subsequently removed by using the Cre/loxP site-specific recombination system of bacteriophage P1. In this manner the same marker could be used more than once in the same background, while the resulting strain could or would remain Anb(R) marker-free. Three plasmids were constructed, each containing a cassette consisting of the Cm(R), Km(R), or Gm(R) gene flanked by two parallel loxP sites and two polylinkers (MCS). To test insertion and excision, cassettes were inserted into the lacZ or galE genes carried on an origamma/pir-dependent suicide plasmid, which contained a dominant Sm(R) gene. The cassettes were crossed into the E. coli genome by homologous recombination (allelic exchange), in a manner analogous to that described by Pósfai et al. [Nucl. Acids Res. 22 (1994) 2392-2398], selecting for the Cm(R), Km(R), or Gm(R), for the LacZ(-) or GalE(-) and for the Sm(S) phenotypes (the latter to assure allelic exchange rather than insertion of the entire plasmid). When required, after selecting the strain with the desired modification, the Cm(R), Km(R), or Gm(R) marker was excised by supplying the Cre function. Cre was provided by the thermosensitive plasmid pJW168, which was transformed into the Anb(R) host at 30 degrees C, and was subsequently eliminated at 42 degrees C. Thus the Anb(R) marker was removed, whereas the lacZ or galE gene remained interrupted by the retained loxP site.

Acetyltransferases↗

PubMed2000

Characterization of the 5' subtilisin (aprE) regulatory region from Bacillus subtilis.

The aprE gene of Bacillus subtilis encodes the major serine alkaline protease known as subtilisin. It is expressed during the transition state and transcribed by the sigma(A) form of the RNA polymerase (RNAP). In this work, we characterized the regulatory region of the aprE gene (rraprE) from B. subtilis. By computer analysis and site-directed mutagenesis, we localized the aprE promoter sequence 7 bp upstream from its transcription initiation site (TIS). We also characterized the static curvature properties of the rraprE DNA and found two different areas of DNA bending, within the first 400 bp upstream of its TIS. We postulate that these particular curved DNA regions could play a role in the interaction with some regulatory proteins and discuss possible implications related to aprE transcription regulation.

Bacillus subtilis↗

PubMed1998

pBRINT-Ts: a plasmid family with a temperature-sensitive replicon, designed for chromosomal integration into the lacZ gene of Escherichia coli.

A pBRINT-Ts family of integrative vectors for Escherichia coli was constructed by using a temperature-sensitive replicon derived from pSC101, a region of homology to the lacZ gene, and various antibiotic resistance markers (kanamycin, chloramphenicol and gentamycin) for selection of the integrants. The gene or group of genes to be integrated can be inserted into a multiple cloning site, flanked by an antibiotic resistance marker and lacZ sequences. A simple and rapid procedure was developed for the selection of cells where allelic exchange had occurred. With this procedure, the efficiency of integration of around 10-3 was observed, using several E. coli strains. From colonies with an integrated pBRINT-Ts plasmid, we detected an average allelic exchange event frequency of 7.5%. As a test for this system, we integrated the amy gene that codes for the alpha-amylase from B. stearothermophilus, into the lacZ gene of E. coli W3110. Production of alpha-amylase was found to be proportional to copy number; at up to 10 copies per chromosome.

Chromosomes, Bacterial↗

PubMed1997

Origin of respiratory sinus arrhythmia in conscious humans. An important role for arterial carotid baroreceptors.

BACKGROUND: We investigated whether respiratory sinus arrhythmia (RSA) in healthy humans originated from central neuronal oscillations or from peripheral baroreceptors responding to respiratory changes in venous return. METHODS AND RESULTS: During subjects' controlled breathing we used sinusoidal neck suction to influence RSA (spectral analysis of RR interval). In 11 subjects, 20-second apnea greatly reduced RSA, which was restored by neck suction at the frequency of respiration. Counteracting the respiration-induced cycles of carotid blood pressure decreased RSA in 13 subjects (from 2136 +/- 682 to 1372 +/- 561 ms2, P < .01). The critical phase of this neck suction was constant for each subject at around the phase shift (with regard to respiration-related fluctuations of blood pressure) best for smoothing respiratory (mechanical) changes in blood pressure. Suction of a non-baroreceptor area (the thigh) did not affect RSA. In 4 subjects, to separate the effects of peripheral baroreceptor afferents from respiration-entrained central oscillation (15 breaths/min), we cycled the neck suction at 12 cycles/min. Increasing neck suction from -7 to -30 mm Hg increased the ratio of the power of the 12 cycles compared with the 15-cycle RSA oscillation in RR interval spectral analysis from 0.26 to 2.57. A 12-cycle/min suction of an area other than the neck had little effect on the RR interval spectrum. CONCLUSIONS: RSA can be mimicked or reduced by stimulation of arterial baroreceptors with cycles of appropriately phased neck suction at the frequency of respiration. This suggests an important influence of the arterial baroreceptors in the generation of RSA in conscious humans.

Adult↗

PubMed1997

Effects of the sinR and degU32 (Hy) mutations on the regulation of the aprE gene in Bacillus subtilis.

The aprE gene of Bacillus subtilis codes for the serine alkaline protease known as subtilisin. Its expression is regulated by a complex network of activators and repressors that includes the products of hpr, degU and sinR. In order to understand the effect of these gene products on subtilisin expression, strains carrying combinations of the degU32(Hy), hpr2 and sinR null mutations, were constructed. We found that in all the genetic backgrounds tested, the sinR null mutation decreased aprE expression. Also, by measuring alkaline phosphatase synthesis and the formation of heat-resistant spores, as indicators of sporulation, we found that some of the mutant strains showed alterations in the sporulation process. These results suggest that these alterations are partially responsible for some of the observed changes in aprE expression.

Bacillus subtilis↗

PubMed1997

Improvement of culture conditions to overproduce beta-galactosidase from Escherichia coli in Bacillus subtilis.

The effect of some culture variables in the production of beta-galactosidase from Escherichia coli in Bacillus subtilis was evaluated. The lacZ gene was expressed in B. subtilis using the regulatory region of the subtilisin gene aprE. The host contained also the hpr2 and degU32 mutations, which are known to overexpress the aprE gene. We found that, when this overproducing B. subtilis strain was grown in mineral medium supplemented with glucose (MMG), beta-galactosidase production was partially growth-associated, as 40%-60% of the maximum enzyme activity was produced before the onset of the stationary phase. In contrast, when a complex medium was used, beta-galactosidase was produced only at low levels during vegetative growth, whereas it accumulated to high levels during early stationary phase. Compared with the results obtained in complex media, a 20% increase in specific beta-galactosidase activity in MMG supplemented with 11.6 g/l glucose was obtained. On the 1-1 fermenter scale, a three-fold increase in volumetric beta-galactosidase activity was obtained when the glucose concentration was varied from 11 g/l to 26 g/l. In addition, glucose feeding during the stationary phase resulted in a twofold increase in volumetric enzyme activity as cellular lysis was prevented. Finally, we showed that oxygen uptake and carbon dioxide evolution rates can be used for on-line determination of the onset of stationary phase, glucose depletion and biomass concentration.

Bacillus subtilis↗

PubMed1997

Lack of peripheral modulation of cardiovascular central oscillatory autonomic activity during apnea in humans.

Respiratory sinus arrhythmia (RSA) high-frequency oscillations (HF) and slow fluctuations in heart rate (LF) are thought to result from entrainment of a medullary oscillator, from the baroreflex, or from a combination of both central and baroreflex mechanisms. We sought to distinguish between the alternatives by examining with spectral analysis the behavior of heart rate (R-R interval) and blood pressure in 10 healthy subjects (mean age 27 +/- 1 yr) during apnea, altering the rate of preapnea entrainment stimuli by changing the frequency either of respiration (controlled at 0.1 or 0.25 Hz) or of baroreceptor stimulation by sinusoidal neck suction (0 to -30 mmHg, 0.1 or 0.2 Hz). During apnea the RSA-EF power decreased (from 6.73 +/- 0.15 to 3.67 +/- 0.10 In ms2: P < 0.0001), regardless of preapnea conditions, whereas LF power was reduced only if preceded by 0.1-Hz respiration or neck suction [from 8.71 +/- 0.18 to 6.52 +/- 0.11 In ms2 (P < 0.001) and from 8.31 +/- 0.23 to 6.90 +/- 0.38 In ms2 (P < 0.01), respectively]. The LF frequency seen in the R-R interval during apnea was slower than the spontaneous LF during 0.25-Hz breathing (0.082 +/- 0.01 vs. 0.112 +/- 0.001 Hz, P < 0.001), but the maneuvers during preapnea had no influence on the observed frequency or other characteristics of the slow oscillations during apnea. Moreover, we found no evidence of a progressive decrease in the power of the oscillation during apnea. The same behavior was observed on the mean blood pressure signal. In conclusion, a slow rhythm is present during apnea. In healthy subjects at rest the characteristics of this oscillation indicate that it could be generated by a central oscillator this may thus contribute to the origin of LF present during normal respiration, in addition to the baroreflex.

Administration, Inhalation↗

PubMed1997

The effect of feeding glucose water to breastfeeding newborns on weight, body temperature, blood glucose, and breastfeeding duration.

In order to determine the effect of feeding glucose water on breastfeeding newborns, we randomly distributed 180 normal newborns into two groups: a glucose water group (GW), fed 5% glucose solution during the first 3 days of life in addition to being breastfed; and an exclusively breastfed nonglucose water group (NGW). The following data were evaluated: weight at 6, 12, 24, 48, and 72 hours of life; temperature during the first 72 hours of life; serum glucose level at 6, 12, 24, and 48 hours; total duration of breastfeeding and age at introduction of infant formulas. In the NGW, there was a greater weight loss at 48 hours but not at 72 hours, temperatures higher than 37.5 degrees C were more frequent, and the mean serum glucose levels at 6, 12, and 24 hours were lower. This group also had more serum glucose level determinations under 2.2 mmol/l (40 mg/dL). However, no infants exhibited hypoglycemic symptoms. Infants in the GW received twice as many formulas during the first month and had a shorter duration of any breastfeeding. Our results suggest that the suppression of feedings with glucose water in the first days of life increases the probability of successful breastfeeding. However, infants who do not receive glucose water in the first few days of life may require greater supervision and close monitoring of blood glucose and body temperature, particularly in the first 24 hours of life.

Adult↗

PubMed1996

A pBRINT family of plasmids for integration of cloned DNA into the Escherichia coli chromosome.

Plasmid pBRINT is an efficient vector for chromosomal integration of cloned DNA into the lacZ gene of Escherichia coli [Balbás et al., Gene 136(1993) 211-213]. A family of related plasmids containing different antibiotic-resistance markers (CmR or GmR or KmR) and a larger multiple cloning site (MCS) has been constructed. This set of plasmids, whose integration efficiencies are as good as those obtained with the prototype plasmid pBRINT, constitutes a collection of tools that allow rapid and easy integration of cloned DNA, at the chromosomal level. Their functionality as integration vectors has been ascertained by integrating the Vitreoscilla sp. hemoglobin-encoding gene and the Photobacterium leiognathi lux genes. To evaluate the level of expression obtained after chromosomal integration, we constructed strains carrying one or two copies of the cat gene integrated in the chromosome, and compared their enzymatic activities with those obtained from a strain carrying cat on a multicopy plasmid.

Base Sequence↗

PubMed1996

A functional Spo0A is required for maximal aprE expression in Bacillus subtilis.

The initiation of sporulation in Bacillus subtilis is under control of the transcriptional factor Spo0A. Most Spo0A mutants fail to initiate the sporulation process and all the sporulation initiated processes such as the synthesis of subtilisin. However, the product of spo0A9V, one of the several spo0A mutants characterized, distinguishes itself in the fact that, while it appears to effectively repress abrB, it fails to activate the spoIIA operon. The aim of this study was to examine the effect of the spo0A9V mutation on aprE expression and we found that in different genetic backgrounds, the spo0A9V mutation has a negative effect on aprE::lacZ expression.

Bacillus subtilis↗