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Biomedical subjects

J M Calvo

Publications and source records attributed to J M Calvo.

At least 19 recordsLinked to original sources
PubMed1997

[Access to bone marrow transplantation in acute myeloblastic leukemia. Study of 52 patients treated in a single center].

BACKGROUND: A single-center experience review about accessibility to bone marrow transplantation (BMT) as postremission therapy for acute myeloid leukemia (AML) is analyzed. PATIENTS AND METHODS: From January 1988 to December 1994, 86 patients were diagnosed from de novo AML in our institution. A BMT was the treatment of choice for all patients younger than 55 years. An allogenic BMT (Allo-BMT) was offered for all patients younger than 35 years with a compatible sibling donor or those older patients, 35-55 years, with bad prognosis features. An autologus BMT (ABMT) was offered to those patients older than 35 years or those younger than 35 without an histocompatible donor. RESULTS: 52 out of 86 diagnosed patients were younger than 50 years (60%). 29 of them were candidates to Allo-BMT (24 patients younger than 35 years and 5 patients older than 35 with refractory disease) and the rest 23 to ABMT. 22 out of the 24 candidates to Allo-BMT entered complete remission (CR) and 12 of them had an HLA-identical donor. The Allo-BMT was performed in CR1 in 7 patients in CR2 in three patients and with refractory disease in two cases. An ABMT was finally planned in 30 patients, 18 patients older than 35 who entered CR and the rest 12 patients younger than 35 years in CR without a sibling donor. Only 11 out of this 30 patients underwent an ABMT in first CR. Reasons for this low number were: early relapse (B), toxicity (6), refuse (2), lost of follow-up (2) and suicide (1). Five out of this early relapse patients underwent an ABMT in CR2. Disease-free survival (DFS) at three years was 23 +/- 10% for the 52 patients included in the study. DFS obtained with Allo-BMT and AMBT were 39 +/- 16% and 63 +/- 22% respectively. CONCLUSIONS: In spite of the new postremission treatment modalities available for AML the rate of longer survivals are still low. When data from BMT is analyzed we must be awared because only a small fraction of patients assigned to BMT will finally access to this treatment.

Adolescent↗
PubMed1996

The leucine-responsive regulatory protein (Lrp) from Escherichia coli. Stoichiometry and minimal requirements for binding to DNA.

Lrp (Leucine-responsive regulatory protein) regulates the expression of a number of operons in Escherichia coli. A recent study of DNA sequences recognized by Lrp established the consensus as a 15-bp sequence, YAGHAWATTWTDCTR (Y = C/T, H = "not G," W = A/T, D ="not C," R = A/G) (Cui, Y., Wang, Q., Stormo, G. D., and Calvo, J. M. (1995) J. Bacteriol. 177, 4872-4880). Here we report the stoichiometry of Lrp binding (an Lrp dimer binds to a single binding site) and studies that define the minimal length of DNA required for binding. A double-stranded 15 mer having a sequence that closely matches the consensus does not show measurable binding to Lrp. One or two base pairs of DNA flanking each end are not sufficient for binding, but constructs having 3-5 additional base pairs (21 mer) show relatively strong binding. Single-stranded flanking DNA also contributes to strong binding. The extent of the contribution to binding is dependent upon whether the single strand is on the left or right of the double-stranded region and whether the polarity of the single-stranded DNA is 5' to 3' or 3' to 5'.

Amino Acid Sequence↗
PubMed1996

Prolonged enhancement of REM sleep produced by carbachol microinjection into the amygdala.

The effect on sleep organization of carbachol microinjected into different amygdaloid nuclei was analysed in 12 cats. Single carbachol doses of 8 micrograms in 0.50 microliter saline were delivered unilaterally or bilaterally into the central, basal, lateral or basolateral amygdaloid nucleus. Carbachol administration into the central nucleus induced a prolonged (5 days) enhancement of both REM sleep and its preceeding slow wave sleep episodes with PGO waves (sommeil phasique a ondes lentes, SPHOL), which was more pronounced following bilateral than unilateral carbachol administration. However, neither SPHOL nor REM sleep changes were produced by administration of carbachol into the other amygdaloid nuclei. We conclude that cholinergic activation of the central amygdaloid nucleus produces a long-term facilitation of REM sleep occurrence.

Amygdala↗
PubMed1996

Effects of nutrition and growth rate on Lrp levels in Escherichia coli.

Lrp (leucine-responsive regulatory protein) activates some Escherichia coli operons that function in anabolism and represses others involved in catabolism (for a review, see J. M. Calvo and R. G. Matthews, Microbiol. Rev. 58:466-490, 1994). This overall pattern suggests that Lrp may help cells adapt to changes in the nutritional environment. Here, we tested the idea that the nutritional richness of the medium determines the amount of Lrp in cells. Lrp was measured directly by Western blotting (immunoblotting) in cells grown in a chemically defined rich medium or in a minimal medium. In addition, transcription from the lrp promoter was assessed with a lacZ reporter gene. The results with these two different measurements were nearly the same, indicating that under the conditions employed, beta-galactosidase measurements can accurately reflect Lrp levels. For cells in a minimal medium, Lrp levels were consistently lowest during the logarithmic phase of growth, but overall, there was not much variation in levels as a function of growth phase (1.3-fold difference between highest and lowest values). However, for cells in a rich medium, Lrp levels dropped 3- to 4-fold during the lag phase, remained constant during the log phase, and then rose to starting levels upon entry into the stationary phase. When cells in the log phase were compared, Lrp levels were 3- to 4-fold higher in cells growing in a minimal medium than those in a rich medium. The levels of lrp expression were the same or slightly higher in strains containing mutations in rpoS, cya, or crp compared with wild-type strains, suggesting that neither RpoS nor the cyclic AMP (cAMP) receptor protein-cAMP complex is required for expression. On the other hand, lrp expression was severely restricted in cells that could not make ppGpp because of mutations in relA and spoT. The reduced expression of lrp during logarithmic growth in a rich medium may be due to low ppGpp levels under these conditions. The repressive effects of rich medium and the stimulatory effects of ppGpp were also observed with a construct having only a minimal lrp promoter (-57 to +21). The results of other experiments suggest that Lrp levels vary inversely with the growth rate of cells instead of being determined by some component of the medium.

Adenylyl Cyclases↗
PubMed1995

A consensus sequence for binding of Lrp to DNA.

Lrp (leucine-responsive regulatory protein) is a major regulatory protein involved in the expression of numerous operons in Escherichia coli. For ilvIH, one of the operons positively regulated by Lrp, Lrp binds to multiple sites upstream of the transcriptional start site and activates transcription. An alignment of 12 Lrp binding sites within ilvIH DNA from two different organisms revealed a tentative consensus sequence AGAAT TTTATTCT (Q. Wang, M. Sacco, E. Ricca, C.T. Lago, M. DeFelice, and J.M. Calvo, Mol. Microbiol. 7:883-891, 1993). To further characterize the binding specificity of Lrp, we used a variation of the Selex procedure of C. Tuerk and L. Gold (Science 249:505-510, 1990) to identify sequences that bound Lrp out of a pool of 10(12) different DNA molecules. We identified 63 related DNA sequences that bound Lrp and estimated their relative binding affinities for Lrp. A consensus sequence derived from analysis of these sequences, YAGHAWATTWT DCTR, where Y = C or T, H = not G, W = A or T, D = not C, and R = A or G, contains clear dyad symmetry and is very similar to the one defined earlier. To test the idea that Lrp in the presence of leucine might bind to a different subset of DNA sequences, we carried out a second selection experiment with leucine present during the binding reactions. DNA sequences selected in the presence or absence of leucine were similar, and leucine did not stimulate binding to any of the sequences that were selected in the presence of leucine. Therefore, it is unlikely that leucine changes the specificity of Lrp binding.

Bacterial Proteins↗
PubMed1995

The amino acid sequence of Lrp is highly conserved in four enteric microorganisms.

Lrp (leucine-responsive regulatory protein) is a global regulator of metabolism in Escherichia coli (J. M. Calvo and R. G. Matthews, Microbiol. Rev. 58:466-490, 1994). The lrp genes from three other enteric microorganisms, Enterobacter aerogenes, Klebsiella aerogenes, and Salmonella typhimurium, were cloned and sequenced. An analysis of these sequences and of the previously determined sequence from E. coli indicated that the vast majority of changes were synonymous rather than nonsynonymous changes. Nucleotide changes occurred at 89 of 492 positions but resulted in amino acid changes at only 2 of 164 positions. This analysis suggests that the Lrp amino acid sequence is highly adapted for function and that almost all amino acid changes lead to a protein that functions less well than the wild-type protein.

Amino Acid Sequence↗
PubMed1994

Sequence determinants of DNA bending in the ilvlH promoter and regulatory region of Escherichia coli.

Previous studies have shown that the promoter/regulatory region of the ilvlH operon displays intrinsic curvature, with the bend center located at position -120 relative to the transcription start site. In this report, a 57 bp sequence spanning the bend center was mutagenized in vitro in order to study the relationship between nucleotide sequence and curvature measured by electrophoresis. The strategy used for analyzing the results consisted of determining the strengths of the relationships between electrophoretic anomaly and predicted curvature calculated by computer programs that differ in wedge angle composition. The results revealed that programs which assume that bending occurs only at AA/TT display good predictive value, with correlation coefficients between electrophoretic anomaly and predicted curvature as high as 0.93. In contrast, a program which assumes that bending occurs at all 16 dinucleotide steps exhibited lower predictive value, while there were no significant relationships between the experimental data and curvature calculated by a program that was based on all non-AA/TT wedge values. These results show that the complete wedge model which incorporates values for all dinucleotide steps does not adequately describe the electrophoretic data in this report.

Acetolactate Synthase↗
PubMed1994

Transitory and short-duration changes of sleep organization provoked by amygdaloid kindling in the cat: a daily 23-hour sleep recording study.

The effect of amygdaloid kindling on sleep organization was tested in chronically implanted cats on a 12:12 light-dark cycle. Electrical kindling stimuli were delivered to the amygdala every 2 h during the light period. Total sleep time and percentage, mean duration, and mean number, as well as the hourly accumulation of waking (W) and sleep (slow wave sleep I and II, and paradoxical sleep) episodes were assessed from daily 23-h sleep recordings, consisting of baseline control recordings, control recordings where cats were briefly alerted at 2-h intervals to mimic the kindling trials manipulation, and recordings on each kindling day. The kindling process was completed within six days achieving three to five consecutive fully kindled seizures. Analysis of total recording time showed that the kindling process enhanced W stage and diminished total sleep time only on the second kindling day. Analysis of light and dark periods demonstrated compensatory W and sleep changes during the dark periods. Hourly sleep stage time accumulation was slowed during kindling trials, but compensated while stimuli were suspended. All of these changes returned to baseline values at the end of kindling. No significant differences between changes induced by kindling and those produced by alerting trials were found. We conclude that sleep alterations can not be exclusively attributed to kindling development, and that sleep is able to become adapted to the focal and generalized seizures effect.

Amygdala↗
PubMed1994

Regulation of the Escherichia coli lrp gene.

Lrp (leucine-responsive regulatory protein) is a major Escherichia coli regulatory protein which regulates expression of a number of operons, some negatively and some positively. This work relates to a characterization of lrp, the gene encoding Lrp. Nucleotide sequencing established that the coding regions of lrp and trxB (encoding thioredoxin reductase) are separated by 543 bp and that the two genes are transcribed in opposite directions. In addition, we used primer extension, deletion analyses, and lrp-lacZ transcriptional fusions to delineate the promoter and regulatory region of the lrp operon. The lrp promoter is located 267 nucleotides upstream of the translational start codon of the lrp gene. In comparison with a wild-type strain, expression of the lrp operon was increased about 3-fold in a strain lacking Lrp and decreased about 10-fold in a strain overproducing Lrp. As observed from DNA mobility shift and DNase I footprinting analyses, Lrp binds to one or more sites within the region -80 to -32 relative to the start point of lrp transcription. A mutational analysis indicated that this same region is at least partly required for repression of lrp expression in vivo. These results demonstrate that autogenous regulation of lrp involves Lrp acting directly to cause repression of lrp transcription.

Bacterial Proteins↗
PubMed1994

The leucine-responsive regulatory protein, a global regulator of metabolism in Escherichia coli.

The leucine-responsive regulatory protein (Lrp) regulates the expression of more than 40 genes and proteins in Escherichia coli. Among the operons that are positively regulated by Lrp are operons involved in amino acid biosynthesis (ilvIH, serA)), in the biosynthesis of pili (pap, fan, fim), and in the assimilation of ammonia (glnA, gltBD). Negatively regulated operons include operons involved in amino acid catabolism (sdaA, tdh) and peptide transport (opp) and the operon coding for Lrp itself (lrp). Detailed studies of a few members of the regulon have shown that Lrp can act directly to activate or repress transcription of target operons. A substantial fraction of operons regulated by Lrp are also regulated by leucine, and the effect of leucine on expression of these operons requires a functional Lrp protein. The patterns of regulation are surprising and interesting: in some cases activation or repression mediated by Lrp is antagonized by leucine, in other cases Lrp-mediated activation or repression is potentiated by leucine, and in still other cases leucine has no effect on Lrp-mediated regulation. Current research is just beginning to elucidate the detailed mechanisms by which Lrp can mediate such a broad spectrum of regulatory effects. Our view of the role of Lrp in metabolism may change as more members of the regulon are identified and their regulation characterized, but at this point Lrp seems to be important in regulating nitrogen metabolism and one-carbon metabolism, permitting adaptations to feast and to famine.

Amino Acid Sequence↗
PubMed1993

Lrp, a global regulatory protein of Escherichia coli, binds co-operatively to multiple sites and activates transcription of ilvIH.

Lrp (Leucine-responsive regulatory protein) has recently been recognized as a major regulatory protein that controls the expression of many operons in Escherichia coli. Footprinting and gel retardation experiments with DNA from ilvIH, one of the operons controlled positively by Lrp, indicate that Lrp binds to six sites over a 200 base-pair region upstream from the promoter. Binding of Lrp to some of these sites is highly co-operative. We suggest a consensus sequence for Lrp binding based upon a comparison of six binding sites. An analysis of mutants indicates that five out of six binding sites are important for transcription activation and that two or three adjacent Lrp binding sites act synergistically in vivo. The observed synergistic effects in vivo may result from co-operative binding of Lrp to adjacent sites. We propose a model in which multiple binding sites contribute to the formation of a nucleoprotein complex, but only a particular proximal site positions Lrp properly so that it interacts with RNA polymerase.

Acetolactate Synthase↗
PubMed1993

Lrp, a major regulatory protein in Escherichia coli, bends DNA and can organize the assembly of a higher-order nucleoprotein structure.

Lrp (Leucine-responsive regulatory protein) is a global regulatory protein that controls the expression of many operons in Escherichia coli. One of those operons, ilvIH, contains six Lrp binding sites located within a several hundred base pair region upstream of the promoter region. Analysis of the binding of Lrp to a set of circularly permuted DNA fragments from this region indicates that Lrp induces DNA bending. The results of DNase I footprinting experiments suggest that Lrp binding to this region facilitates the formation of a higher-order nucleoprotein structure. To define more precisely the degree of bending associated with Lrp binding, one or two binding sites were separately cloned into a pBend vector and analyzed. Lrp induced a bend of approximately 52 degrees upon binding to a single binding site, and the angle of bending is increased to at least 135 degrees when Lrp binds to two adjacent sites. Lrp-induced DNA bending, and a natural sequence-directed bend that exists within ilvIH DNA, may be architectural elements that facilitate the assembly of a nucleoprotein complex.

Bacterial Proteins↗
PubMed1993

Organization of Lrp-binding sites upstream of ilvIH in Salmonella typhimurium.

Lrp, a major regulatory protein in Escherichia coli, controls the expression of numerous operons, including ilvIH. Lrp binds to six sites upstream of ilvIH, and Lrp binding is required for ilvIH expression. We show here that an Lrp-like protein is also present in Salmonella typhimurium. This protein can bind both E. coli and S. typhimurium ilvIH DNA, as can E. coli Lrp. Methidiumpropyl-EDTA footprinting studies were performed with purified E. coli Lrp and S. typhimurium ilvIH DNA. Six binding sites were defined, three of them being similar to corresponding sites in E. coli, and three being organized differently. A consensus derived from six S. typhimurium sites is compatible with that derived from a similar analysis of E. coli sequences.

Acetolactate Synthase↗
PubMed1993

Regions of the Bacillus subtilis ilv-leu operon involved in regulation by leucine.

The ilv-leu operon of Bacillus subtilis is regulated in part by transcription attenuation. The cis-acting elements required for regulation by leucine lie within a 683-bp fragment of DNA from the region upstream of ilvB, the first gene of the operon. This fragment contains the ilv-leu promoter and 482 bp of the ilv-leu leader region. Spontaneous mutations that lead to increased expression of the operon were shown to lie in an imperfect inverted repeat encoding the terminator stem within the leader region. Mutations within the inverted repeat of the terminator destroyed most of the leucine-mediated repression. The remaining leucine-mediated repression probably resulted from a decrease in transcription initiation. A systematic analysis of other deletions within the ilv-leu leader region identified a 40-bp region required for the derepression that occurred during leucine limitation. This region lies within a potential RNA stem-and-loop structure that is probably required for leucine-dependent control. Deletion analysis also suggested that alternate secondary structures proximal to the terminator are involved in allowing transcription to proceed beyond the terminator. Additional experiments suggested that attenuation of the ilv-leu operon is not dependent on coupling translation to transcription of the leader region. Our data support a model proposed by Grundy and Henkin (F. J. Grundy and T. M. Henkin, Cell 74:475-482, 1993) in which uncharged tRNA acts as a positive regulatory factor to increase gene expression during amino acid limitation.

Bacillus subtilis↗
PubMed1993

Mutations affecting the ability of Escherichia coli Lrp to bind DNA, activate transcription, or respond to leucine.

Lrp is a regulatory protein in Escherichia coli that increases expression of some operons and decreases expression of others. Mutations in Lrp were isolated on the basis of their effects on ilvIH, one of the operons regulated positively by Lrp. The ilvIH operon encodes an enzyme involved in the biosynthesis of leucine, valine, and isoleucine, and expression of this operon is repressed when cells are grown in the presence of leucine. Three groups of mutants were isolated. Mutant strains that were resistant to the repressive effects of leucine were termed leucine response mutants. These mutants had changes in the Lrp amino acid sequence between amino acid residues 108 and 149. Mutant strains having low expression of ilvIH in vivo were identified as colonies having reduced expression of a reporter gene. For some of these mutants, called DNA-binding mutants, binding to ilvIH DNA in vitro was markedly reduced. The mutations in these strains caused changes in Lrp between amino acids 16 and 70. Six of ten of these mutations were within a region having a putative helix-turn-helix motif. A third group of mutants had low ilvIH expression in vivo but apparently normal DNA binding in vitro. These mutants were called activation mutants since they affected the ability of Lrp to activate expression. Lrp from these strains had changes in amino acids between residues 76 and 125. This study suggests that Lrp has separate domains responsible for binding DNA, activating transcription, and responding to leucine.

Bacterial Proteins↗