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Biomedical subjects

J C Alonso

Publications and source records attributed to J C Alonso.

At least 19 recordsLinked to original sources
PubMed2004

Diagnosis of orthopedic infection in clinical practice using Tc-99m sulesomab (antigranulocyte monoclonal antibody fragment Fab'2).

OBJECTIVE: The aim of the study was to analyze the use of Tc-99m-labeled antigranulocyte monoclonal antibody fragment Fab'2 (SMab, Leukoscan) in the diagnosis of acute osteomyelitis and articular prosthesis infection. One hundred seventeen patients (118 studies) have been included, 92 women and 25 men. Thirty-seven had a suspicion of osteomyelitis and 81 with suspicion of infection of a hip (35 patients) or a knee (45 patients) prosthesis. The mean age was 64 years. A 3-phase bone scan and a SMab scan were obtained in all patients. Antigranulocyte scans were obtained within 3 and 8 hours postinjection of 20 mCi (740 MBq) of Tc-99m-labeled antibody. Diagnosis of infection was based on bone biopsy in 8 cases and on at least 2 positive cultures in the remaining cases. Infection was ruled out with negative cultures or 1-year patient follow up with negative C-reactive protein and normal erythrocyte sedimentation rate. RESULTS: Sensitivity, specificity, accuracy, and negative predictive value of Tc 99m-SMab scintigraphy were: 75%, 95%, 87%, and 92% for long bone osteomyelitis and 80%, 89%, 87%, and 91% for articular prosthesis infection, respectively. Positive predictive values were 83% and 63%, respectively. CONCLUSION: Tc-99m-antigranulocyte monoclonal antibody fragment Fab'2 scintigraphy seems to be a useful method in the diagnosis of osteoarticular infection.

Acute Disease↗
PubMed2003

Crystallization of the Bacillus subtilis SPP1 bacteriophage helicase loader protein G39P.

The essential helicase loader protein G39P encoded by Bacillus subtilis SPP1 phage has been overproduced in Escherichia coli and purified. The wild-type protein has been crystallized by the hanging-drop vapour-diffusion method in a primitive hexagonal space group, probably P6(1)22/P6(5)22, but the crystals diffract to only 3.4 A and are poorly reproducible. Mass-spectrometric analysis has revealed marked proteolytic cleavage from the C-terminus and the presence of a major species corresponding to deletion of the 14 C-terminal residues. Thus, a new variant of the protein (G39P112) has been engineered that corresponds to a 14-residue C-terminal truncation. The G39P112 variant has also been crystallized but now in a primitive orthorhombic form, probably P2(1)2(1)2 or P2(1)2(1)2(1), with unit-cell parameters a = 85.6, b = 89.7, c = 47.6 A, with diffraction to 2.4 A on a synchrotron source and with greatly improved reproducibility. Calculation of V(M) values for this G39P112 variant suggests the presence of three monomers in the asymmetric unit, corresponding to a solvent content of about 47%. A selenomethionine-incorporated form of the protein has been produced and a full three-wavelength MAD data collection undertaken.

Bacillus subtilis↗
PubMed2001

Flavones inhibit the hexameric replicative helicase RepA.

Helicases couple the hydrolysis of nucleoside triphosphates (NTPs) to the unwinding of double-stranded nucleic acids and are essential in DNA metabolism. Thus far, no inhibitors are known for helicases except heliquinomycin isolated from Streptomyces sp. As the three-dimensional structure of the hexameric replicative DNA helicase RepA encoded by the broad host-range plasmid RSF1010 is known, this protein served as a model helicase to search for inhibitory compounds. The commercially available flavone derivatives luteolin, morin, myricetin and dimyricetin (an oxidation product of myricetin) inhibited the ATPase and double-stranded DNA unwinding activities of RepA. Dimyricetin was the most effective inhibitor for both activities. Single-stranded DNA-dependent RepA ATPase activity is inhibited non-competitively by all four compounds. This finding contrasts the inhibition of phosphoinositide 3-kinase by flavones that fit into the ATP binding pocket of this enzyme. Myricetin also inhibited the growth of a Gram-positive and a Gram-negative bacterial species. As we found other hexameric and non-hexameric prokaryotic helicases to be differentially sensitive to myricetin, flavones may provide substructures for the design of molecules helpful for unraveling the mechanism of helicase action and of novel pharmacologically useful molecules.

Adenosine Triphosphatases↗
PubMed2001

Crystal structure of omega transcriptional repressor encoded by Streptococcus pyogenes plasmid pSM19035 at 1.5 A resolution.

The 71 amino acid residue omega protein encoded by the Streptococcus pyogenes non-conjugative plasmid pSM19035 is a transcriptional repressor that regulates expression of genes for copy number control and stable maintenance of plasmids. The crystal structure of omega protein has been determined by multiple isomorphous replacement, including anomalous scattering and refined to an R-factor of 21.1 % (R(free)=23.2 %) at 1.5 A resolution. Two monomers related by a non-crystallographic 2-fold axis form a homodimer that occupies the asymmetric unit. Each polypeptide chain is folded into two alpha-helices and one beta-strand forming an antiparallel beta-ribbon in the homodimer. The N-terminal regions (1-23 and 1-22 in subunits I and II, respectively) are not defined in the electron density due to proteolysis of the N-terminal 20 amino acid residues during crystallisation and partial disorder. The omega protein belongs to the structural superfamily of MetJ/Arc repressors featuring a ribbon-helix-helix DNA-binding motif with the beta-ribbon located in and recognizing the major groove of operator DNA; according to a modelled omega protein-DNA complex, residues Arg31 and Arg31' on the beta-ribbon are in positions to interact with a nucleobase, especially guanine.

Amino Acid Sequence↗
PubMed2001

Effect of the recU suppressors sms and subA on DNA repair and homologous recombination in Bacillus subtilis.

In Bacillus subtilis, mutant alleles of the genes sms and subA partially suppress the recombination phenotype of recU cells. When present in an otherwise Rec(+) strain, Delta sms and Delta subA alleles render cells slightly sensitive to DNA-damaging agents, and impair chromosomal transformation (3- to 10-fold reduction), but do not affect plasmid transformation (less than 1.5-fold reduction). The Delta sms and Delta subA alleles were introduced into rec-deficient strains representative of the epistatic groups alpha (recF strain), beta (addA addB), gamma (recH), epsilon (recB, Delta recU and recD strains) and zeta (Delta recS). Both the Delta sms and Delta subA mutations were found to increase sensitivity to DNA-damaging agents in recF, Delta recS and addAB cells. In contrast, the Delta sms mutations decreased the sensitivity of recB, Delta recU, recD and recH cells, and the Delta subA mutation decreased the sensitivity of recB and Delta recU cells to DNA-damaging agents. Functions classified within the epistatic groups alpha, epsilon and zeta are required for intramolecular recombination, measured as plasmid transformation. The Delta sms and Delta subA mutations, which partially suppressed the recombinational repair phenotype of mutants for functions within epistatic group epsilon, enhanced plasmid transformation of recU (recB, recD) and recS cells by 10- to 20-fold. In the absence of the proteins Sms and SubA, the recombination machinery is apparently redirected towards (an) alternative pathway(s). Furthermore, the shared ability of the Delta sms and Delta subA mutations to act as indirect suppressors of recB, recU and recD mutations supports the classification of the recBUD genes within epistatic group epsilon.

Alleles↗
PubMed2001

Stability and DNA-binding properties of the omega regulator protein from the broad-host range Streptococcus pyogenes plasmid pSM19035.

At the transcriptional level, the pSM19035-encoded omega protein coordinates the expression of proteins required for control of copy number and maintenance of plasmids. Using circular dichroism, fluorescence spectroscopy, ultracentrifugation and an electrophoretic mobility shift assay, the wild-type omega protein and a variant with a C-terminal hexa-histidine tag (omega-H(6)) were characterized. The omega protein is mainly alpha-helical (42%), occurs as homodimer in solution, unfolds thermally with half transition temperatures, T(m), between approximately 43 and approximately 78 degrees C depending on the ionic strength of the buffer, and binds PcopS-DNA with high affinity. The omega-H(6) protein has a modified conformation with lower alpha-helix content (29%), lower thermal stability, and strongly reduced affinity to PcopS-DNA.

Bacterial Proteins↗
PubMed2001

Glu-256 is a main structural determinant for oligomerisation of human arginase I.

One determinant that could play a role in the quaternary structure of human arginase is the pair of salt links between the strictly conserved residues R255 from one monomer and E256 from every adjacent subunit. In this work, the ionic interaction between monomers was disrupted by expressing a human arginase where Glu-256 had been substituted by Gln. Biochemical analyses of the mutant protein showed that: (i) it shares the wild-type kinetic parameters of the arginine substrate; (ii) E256Q arginase behaves as a monomer by gel filtration; (iii) it is drastically inactivated by dialysis in the presence of EDTA, an inhibitory effect which is reversed by addition of Mn(2+); and (iv) the mutant enzyme loses thermal stability. The lack of oligomerisation for E256Q arginase and the conservation of E256 throughout evolution of the protein family suggest that this residue is involved in the quaternary structure of arginases.

Amino Acid Sequence↗
PubMed2001

Crystallization and preliminary X-ray diffraction studies of the epsilonzeta addiction system encoded by Streptococcus pyogenes plasmid pSM19035.

The proteins encoded by the Streptococcus pyogenes broad-host range and low copy-number plasmid pSM19035 form a toxin-antitoxin module that secures stable maintenance by causing the death of plasmid-free segregants. The epsilonzeta protein complex was crystallized in four different forms at pH 5.0 and pH 7.0 using the vapour-diffusion method with PEG 3350 and ethylene glycol as precipitants. Three of the crystal forms were obtained in the same droplet under identical conditions at pH 5.0. One form belongs to the enantiomorphic space groups P4(3)2(1)2 or P4(1)2(1)2. For the other two, the X-ray reflection conditions match those of space group P2(1)2(1)2(1), one representing a superlattice of the other. A crystal form growing at pH 7.0 also belongs to space group P2(1)2(1)2(1), but there is no indication of a structural relationship to the other orthorhombic forms. Initially, the crystals diffracted to 2.9 A resolution and diffracted to 1.95 A after soaking at pH 7.0. A preparation of selenomethionyl epsilonzeta protein complex yielded single crystals suitable for X-ray diffraction experiments using synchrotron sources.

Bacterial Proteins↗
PubMed2001

New insights into host factor requirements for prokaryotic beta-recombinase-mediated reactions in mammalian cells.

The prokaryotic beta-recombinase catalyzes site-specific recombination between two directly oriented minimal six sites in mammalian cells, both on episomic and chromatin-integrated substrates. Using a specific recombination activated gene expression system, we report the site-specific recombination activity of an enhanced green fluorescent protein (EGFP) fused version of beta-recombinase (beta-EGFP). This allows expression of active beta-recombinase detectable in vivo and in fixed cells by fluorescence microscopy. In addition, cellular viability is compatible with a substantial level of expression of the beta-EGFP protein. Using fluorescence-activated cell sorting, we have been able to enrich cell populations expressing this fusion protein. Application of this strategy has allowed us to study in more depth the host factor requirements for this system. Previous work showed that eukaryotic HMG1 protein was necessary and sufficient to help beta-recombinase activity in vitro. The influence of ectopic expression of HMG1 protein in the recombination process has been analyzed, indicating that HMG1 overexpression does not lead to a significant increase on the efficiency of beta-recombinase-mediated recombination both on episomal substrates and chromatin-associated targets. In addition, beta-recombinase-mediated recombination has been demonstrated in HMG1 deficient cells at the same levels as in wild type cells. These data demonstrate the existence of cellular factors different from HMG-1 that can act as helpers for beta-recombinase activity in the eukaryotic environment.

3T3 Cells↗
PubMed2001

[Utility of SPECT with 99m Tc-tetrofosmin and stimulus with dipyridamole in patients with permanent ventricular pacemakers].

BACKGROUND AND OBJECTIVE: Patients with permanent ventricular pacemakers (PP) are a difficult subgroup to assess in the study of coronary artery disease. The objective of this study was to evaluate the diagnostic value of the myocardial perfusion scintigraphy with 99mTC-Tetrofosmin (Tc-Tf) and stimulus with dipyridamole in patients with PP and suspected coronary artery disease. PATIENTS AND METHODS: Fourteen patients with suspected coronary artery disease and without structural cardiopathy, who underwent Tc-Tf and cardiac catheterization, were studied retrospectively. Sensitivity, specificity, predictive values and Kappa index were calculated for the diagnosis of coronary artery disease, multivessel disease, and for each one of the coronary arteries (left anterior descending, right coronary, and circumflex). In addition, the correlation between the number of territories with perfusion defects and the number of diseased vessels was studied. RESULTS: In regards to the diagnosis of coronary artery disease, sensitivity, specificity, positive predictive value, negative predictive value and the Kappa index were 100%, 50%, 83%, 100% and 0.55, respectively. For multi-vessel disease, these values were 83%, 64%, 64%, 83% and 0.43 respectively. The correlation coefficient between the number of territories with perfusion defects and the number of diseased vessels was 0.61 (p = 0.02). In the diagnosis of anterior descending disease, sensitivity and specificity were 83% and 88% respectively. For the right coronary artery, these values were 100% and 44% and for the circumflex artery 38% and 83%, respectively. CONCLUSION: In patients with PP and suspected coronary artery disease, myocardial perfusion scintigraphy with Tc-Tf and stimulus with dipyridamole is of great value in the diagnosis of coronary artery disease and in the assessment of its extension.

Aged↗
PubMed2001

Lithium inhibits caspase 3 activation and dephosphorylation of PKB and GSK3 induced by K+ deprivation in cerebellar granule cells.

Lithium protects cerebellar granule cells from apoptosis induced by low potassium, and also from other apoptotic stimuli. However, the precise mechanism by which this occurs is not understood. When cerebellar granule cells were switched to low potassium medium, the activation of caspase 3 was detected within 6 h, suggesting a role of caspase 3 in mediating apoptosis under conditions of low potassium. In the same conditions, lithium (5 mM) inhibited the activation of caspase 3 induced by low potassium. As lithium did not inhibit caspase 3 activity in vitro, these results suggest that this ion inhibits an upstream component that is required for caspase 3 activation. Lithium is known to inhibit a kinase termed glycogen sythase kinase 3 (GSK3), which is implicated in the survival pathway of phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB). Here we demonstrate that low potassium in the absence of lithium induces the dephosphorylation, and therefore the activation, of GSK3. However, when lithium was present, GSK3 remained phosphorylated at the same level as observed under conditions of high potassium. Low potassium induced the dephosphorylation and inactivation of PKB, whereas when lithium was present PKB was not dephosphorylated. Our results allow us to propose a new hypothesis about the action mechanism of lithium, this ion could inhibit a serine-threonine phosphatase induced by potassium deprivation.

Animals↗
PubMed2001

Genetic recombination in Bacillus subtilis 168: effect of DeltahelD on DNA repair and homologous recombination.

The B. subtilis DeltahelD allele rendered cells proficient in transformational recombination and moderately sensitive to methyl methanesulfonate when present in an otherwise Rec(+) strain. The DeltahelD allele was introduced into rec-deficient strains representative of the alpha (recF strain), beta (addA addB), gamma (recH), epsilon (DeltarecU), and zeta (DeltarecS) epistatic groups. The DeltahelD mutation increased the sensitivity to DNA-damaging agents of addAB, DeltarecU, and DeltarecS cells, did not affect the survival of recH cells, and decreased the sensitivity of recF cells. DeltahelD also partially suppressed the DNA repair phenotype of other mutations classified within the alpha epistatic group, namely the recL, DeltarecO, and recR mutations. The DeltahelD allele marginally reduced plasmid transformation (three- to sevenfold) of mutations classified within the alpha, beta, and gamma epistatic groups. Altogether, these data indicate that the loss of helicase IV might stabilize recombination repair intermediates formed in the absence of recFLOR and render recFLOR, addAB, and recH cells impaired in plasmid transformation.

Adenosine Triphosphatases↗
PubMed2000

Functional analysis of the terminase large subunit, G2P, of Bacillus subtilis bacteriophage SPP1.

The terminase of bacteriophage SPP1, constituted by a large (G2P) and a small (G1P) subunit, is essential for the initiation of DNA packaging. A hexa-histidine G2P (H6-G2P), which is functional in vivo, possesses endonuclease, ATPase, and double-stranded DNA binding activities. H6-G2P introduces a cut with preference at the 5'-RCGG downward arrowCW-3' sequence. Distamycin A, which is a minor groove binder that mimics the architectural structure generated by G1P at pac, enhances the specific cut at both bona fide 5'-CTATTGCGG downward arrowC-3' sequences within pacC of SPP1 and SF6 phages. H6-G2P hydrolyzes rATP or dATP to the corresponding rADP or dADP and P(i). H6-G2P interacts with two discrete G1P domains (I and II). Full-length G1P and G1PDeltaN62 (lacking domain I) stimulate 3.5- and 1.9-fold, respectively, the ATPase activity of H6-G2P. The results presented suggest that a DNA structure, artificially promoted by distamycin A or facilitated by the assembly of G1P at pacL and/or pacR, stimulates H6-G2P cleavage at both target sites within pacC. In the presence of two G1P decamers per H6-G2P monomer, the H6-G2P endonuclease is repressed, and the ATPase activity stimulated. Based on these results, we propose a model that can account for the role of terminase in headful packaging.

Adenosine Triphosphatases↗
PubMed2000

Shape and DNA packaging activity of bacteriophage SPP1 procapsid: protein components and interactions during assembly.

The procapsid of the Bacillus subtilis bacteriophage SPP1 is formed by the major capsid protein gp13, the scaffolding protein gp11, the portal protein gp6, and the accessory protein gp7. The protein stoichiometry suggests a T=7 symmetry for the SPP1 procapsid. Overexpression of SPP1 procapsid proteins in Escherichia coli leads to formation of biologically active procapsids, procapsid-like, and aberrant structures. Co-production of gp11, gp13 and gp6 is essential for assembly of procapsids competent for DNA packaging in vitro. Presence of gp7 in the procapsid increases the yield of viable phages assembled during the reaction in vitro five- to tenfold. Formation of closed procapsid-like structures requires uniquely the presence of the major head protein and the scaffolding protein. The two proteins interact only when co-produced but not when mixed in vitro after separate synthesis. Gp11 controls the polymerization of gp13 into normal (T=7) and small sized (T=4?) procapsids. Predominant formation of T=7 procapsids requires presence of the portal protein. This implies that the portal protein has to be integrated at an initial stage of the capsid assembly process. Its presence, however, does not have a detectable effect on the rate of procapsid assembly during SPP1 infection. A stable interaction between gp6 and the two major procapsid proteins was only detected when the three proteins are co-produced. Efficient incorporation of a single portal protein in the procapsid appears to require a structural context created by gp11 and gp13 early during assembly, rather than strong interactions with any of those proteins. Gp7, which binds directly to gp6 both in vivo and in vitro, is not necessary for incorporation of the portal protein in the procapsid structure.

Bacillus↗
PubMed2000

Plasmid copy-number control and better-than-random segregation genes of pSM19035 share a common regulator.

Transcription initiation of the copy-number control and better-than-random segregation genes of the broad-host-range and low-copy-number plasmid pSM19035 are subjected to repression by the autoregulated pSM19035-encoded omega product in Bacillus subtilis cells. The promoters of the copS (Pcop1 and Pcop2), delta (Pdelta), and omega (Pomega) genes have been mapped. These promoters are embedded in a set of either seven copies of a 7-bp direct repeat or in a block consisting of two 7-bp direct repeats and one 7-bp inverted repeat; the blocks are present either two or three times. The cooperative binding of omega protein to the repeats on the Pcop1, Pcop2, Pdelta, and Pomega promoters represses transcription initiation by a mechanism that does not exclude sigma(A)RNAP from the promoters. These results indicate that omega protein regulates plasmid maintenance by controlling the copy number on the one hand and by regulating the amount of proteins required for better-than-random segregation on the other hand.

Bacillus subtilis↗
PubMed2000

[Results of myocardial scintigraphy with 99mTc-tetrofosmin and dipyridamole administration in patients diagnosed of microvascular angina].

The objective of the study was to evaluate the results of the myocardial scintigraphy with 99mTc-tetrofosmin (Tc-Tf) and pharmacological stimulation with dipyridamole in patients diagnosed of microvascular angina. The study population is made up of 50 patients discharged with the diagnosis of microvascular angina who had undergone Tc-Tf-dipyridamole. Clinical and electrical positivity appeared in 40% and 12% of the patients, respectively. Myocardial perfusion defects were found in 35 patients (70%), and were reversible in 21 (60%), fixed in 11 (21%), and combined in 3 (9%). Abnormalities were inferior, anteroseptal and lateral in 21, 18, and 2 patients, respectively. Patients with a positive exercise treadmill test, compared with those with a negative one, had more frequent perfusion abnormalities (91% vs 50%, p = 0.0327) and myocardial ischemia (64% vs 20%, p = 0.392). Women, in comparison with men, had angina (56% vs 22%, p = 0.013), and anteroseptal perfusion abnormalities (26% vs 4%, p = 0.028) more frequently. On the contrary, men had inferior perfusion abnormalities more frequently (57% vs 30%, p = 0.057). Thus, Tc-Tf-dipyridamole shows perfusion abnormalities in 70% of patients with microvascular angina (91% in patients with a positive exercise treadmill test). Scintigraphic pattern may be partially conditioned by gender in these patients.

Coronary Circulation↗
PubMed2000

[Bone scintigraphy findings in patients with recently diagnosed adenocarcinoma of the prostate: relationship with prostate specific antigen levels].

BACKGROUND AND OBJECTIVES: Although it has been suggested that bone scintigraphy (BS) may not be necessary in patients with prostate adenocarcinoma (PA) and normal prostate specific antigen (PSA) plasma levels, controversy still remains. The objective of the study was to evaluate the existing relationship between PSA plasma levels and BS findings in patients with a recently diagnosed PA in order to assess whether BS may be omitted on the basis of the PSA levels in these patients. METHODS: The 475 patients (70+/-7 years old) consecutively diagnosed of PA between 1994 and 1998 in our institution made up the study population. PSA plasmatic levels were determined and BS was performed (body planar study after 99mTc-methyl diphosphonate 900 MBq administration) in all the patients. RESULTS: In 362 patients (76.2%), BS was negative, in 108 (22.7%) positive and undetermined in the remaining 5 patients (1.1%). The mean PSA level in the whole study population was 74+/-267 ng/ml (range 0.4-4.200) and was higher in patients with positive GO (218+/-512 vs 31+/-89, p<0,0001). As PSA increased, the rate of patients with positive BS was significantly higher, this being 0%, 16.4%, 9.7%, 14.0%, 31.0% and 48.3% in patients with PSA 4, 4.1-10; 10.1-20; 20.1-30; 30.1-40, and >40 ng/ml, respectively (p<0.0001). In patients with positive BS, the PSA levels were 20 and 10 ng/ml in 30.6% and 18.6% of the patients, respectively. CONCLUSION: According to our experience, there is a significant association between PSA plasma levels and the BS results in patients with recently diagnosed PA. However, a significant proportion of patients with bone metastasis have normal PSA levels, and therefore BS should be performed in all patients with recently diagnosed PA regardless of the PSA levels.

Aged↗