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Biomedical subjects

J L Ramos

Publications and source records attributed to J L Ramos.

At least 19 recordsLinked to original sources
PubMed2006

Cross validation with the mood disorder questionnaire (MDQ) of an instrument for the detection of hypomania in Spanish: the 32 item hypomania symptom check list (HCL-32).

BACKGROUND: The detection and diagnosis of present or past hypomanic episodes is of key importance for the differential diagnosis between depressive disorders and type II bipolar disorder. However, there are few instruments available to satisfactorily screen for the latter condition. The Hypomania Symptom Checklist-32 (HCL-32) is a self-applied questionnaire with 32 hypomania items and 8 severity and functional impact items which is being developed in several European countries for this purpose. Our aim was to develop and validate the psychometric properties of the HCL-32 scale in Spain in patients with bipolar disorder and to compare its properties with other instruments available for the detection of bipolar II disorder. METHODS: Patients were selected from 15 psychiatric outpatient departments, diagnosed with type I or type II bipolar disorder (BDI and BDII) and unipolar major depression (MD) according to DSM-IV-TR criteria. A control group of healthy subjects (HS) was likewise assessed. The patient selection criteria included a well-established diagnosis and a stable disorder and pharmacological treatment. The HCL-32 was administered to 237 subjects distributed among the above groups, on two occasions four weeks apart. We analysed the internal consistency, test-retest reliability and discriminative capacity of the HCL-32. RESULTS: The internal consistency of the Spanish version of the HCL-32, evaluated by Cronbach's alpha, was 0.94. Mean of affirmative questions by group were 21.2 (SD 5.8) for BDI, 19.3 (SD 6.2) for BDII, 8.6 (SD 6.6) for MD and 6.6 (SD 6.1) for HS, with statistically significant differences between them (Kruskal-Wallis test, p<0.001). Concurrent validity using the diagnosis variable was 0.72. Test-retest reliability was 0.90. We analysed the best cut-off point by means of a ROC curve analysis; for 14 affirmative responses, a sensitivity of 0.85 95%CI (0.78, 0.91) and specificity of 0.79, 95%CI (0.72, 0.87) were obtained. The positive and negative probability ratios were 4.1 and 5.3 (1/0.19 respectively). HCL-32 shows a dual factor structure of items, one as an energy-activity factor and another one as a factor involving items related to disinhibition and problems with self-control and attention. LIMITATIONS: The sample size of bipolar patients (particularly type BDII) should be increased in further studies. CONCLUSIONS: The Spanish version of the HCL-32 has good psychometric properties and sufficient sensitivity and specificity, detecting 8 out of every 10 patients with BD. The HCL-32 is a useful screening tool of patients with bipolar disorder in clinical settings. In its present form it adequately discriminates between bipolar and unipolar or healthy subjects, but not between BD I and BII.

Adult↗
PubMed2005

Hyperhomocysteinemia in patients with mild chronic renal failure.

BACKGROUND: The aim of this study was to evaluate the prevalence of high plasma levels of homocysteine in patients with mild renal failure. METHODS: Forty-six chronic renal failure patients (25 males and 21 females, mean age 55.6+/-14.4 years) were recruited for the study. Mean plasma creatinine was 2.1+/-1.0 mg/dl and mean creatinine clearance was 50.6+/-26.3 ml/min. Patients with severe renal failure were excluded. Patients were compared with a control group with normal renal function (n=35, 22 men and 13 women, mean age 50.0+/-11.5 years). Plasma homocysteine values were measured in both groups at baseline and after an oral overload of methionine. RESULTS: Baseline homocysteine levels of patients were higher than those of controls (16.5+/-7.3 vs. 10.4+/-4.2 micromol/l, p<0.0001). Some 34 patients and 4 controls had increased plasma homocysteine levels at baseline. After the oral overload, 4 more patients had abnormally increased homocysteine levels, meaning that 83% of the patients with chronic renal failure had hyperhomocysteinemia. CONCLUSIONS: Hyperhomocysteinemia is a very common finding among patients with mild renal failure. The need for vitamin supplementation should be evaluated in the first stage of chronic renal failure.

Journal Article↗
PubMed2003

Inhibition of growth and induction of apoptosis in human breast cancer by transfection of gef gene.

The gef gene has cell-killing functions in Escherichia coli. To evaluate the feasibility of using this gene as a new strategy for cancer therapy, we transfected it in MCF-7 cells derived from breast cancer (MCF-7TG). The gef gene was cloned in a pMAMneo vector under the control of a mouse mammary tumour virus promoter, inducible by dexamethasone (Dex), and was transfected with liposomes. After selection and induction, expression of the gef gene was confirmed by reverse transcription-polymerase chain reactions (RT-PCR) and Western blot. Cell viability was determined with a haemocytometre and the sulphorodamine B colorimetric assay, and the cell cycle was studied by propidium iodide (PI) staining. Annexin V-FITC and PI assays were used to evaluate apoptosis, which was confirmed by electron microscopy. In comparison with MCF-7 parental cells and MCF-7 cells transfected with an empty vector, MCF-7TG cells induced with Dex showed a significant decrease in proliferation rate, which was associated with evidence of apoptosis. Morphological findings confirmed apoptosis and showed a typical pattern of mitochondrial dilation. Furthermore, the cell cycle was characterised by premature progression from G(1) to S phase and G(2) delay. Our results show that the gef gene was able to decrease proliferation in a breast cancer cell line, and induce apoptosis. These findings suggest that the gef gene is a potential candidate for tumour therapy.

Animals↗
PubMed2002

Analysis of polyethylene thickness of tibial components in total knee replacement.

BACKGROUND: Excessive wear of the polyethylene bearing surfaces of tibial components has become an important factor in early failure of total knee arthroplasty. Inadequate thickness of the polyethylene insert is one cause of excessive wear, and various minimum thicknesses have been recommended in order to reduce contact stresses within the polyethylene. However, the true thicknesses of modular polyethylene tibial inserts typically are not stated accurately by the manufacturers in their product information. The purpose of this study was to determine whether the information about the thickness of tibial inserts supplied by the manufacturers is adequate. METHODS: Five of the thinnest available polyethylene tibial inserts from five different manufacturers were selected. The minimum thickness of each was measured with use of a Sigma electronic micrometer comparator to an accuracy of +/-0.005 mm. RESULTS: The stated thicknesses of the polyethylene tibial inserts were 8, 9, and 10 mm, values that differed markedly from the actual minimum thicknesses, which ranged from 5.5722 to 6.2048 mm (mean values). CONCLUSION: The thickness of polyethylene tibial inserts has been implicated as a potential cause of excessive wear and early failure of total knee replacements. This paper highlights the fact that the information supplied by the manufacturers is inaccurate and potentially misleading; in one case, the true thickness was much less than the recommended minimum thickness. We recommend that the minimum thickness of the tibial components as well as the combined thickness of the polyethylene insert and the metal tibial tray be specified in the product information and on the packaged insert.

Arthroplasty, Replacement, Knee↗
PubMed2001

Residues 137 and 153 of XylS influence contacts with the C-terminal domain of the RNA polymerase alpha subunit.

XylS and XylS1 are transcriptional regulators that stimulate transcription from the Pm promoter for the meta-cleavage pathway operon for alkylbenzoate degradation. These regulators that differ in five amino acids interact with alpha-CTD domain of RNA polymerase. These interactions take place preferentially through residues 291 in XylS and 289 in XylS1. Substitution at position 137 and 153 in XylS influence the interactions with alpha-CTD because single and double mutants in these positions turned preferential interactions to residue 289.

Bacterial Proteins↗
PubMed2001

Interactions of the XylS regulators with the C-terminal domain of the RNA polymerase alpha subunit influence the expression level from the cognate Pm promoter.

The Pseudomonas putida meta-cleavage operon encodes the enzymes for the catabolism of alkylbenzoates. Activation of meta-operon transcription is mediated by the XylS protein which, upon activation by effectors, binds two sites between -70 and -35 with respect to the main transcription initiation point at the Pm promoter. Two naturally occurring regulators, XylS and XylS1, that differ by only five amino acids, have been analyzed with regard to potential interactions of these positive regulators with the C-terminal domain of the alpha subunit of RNA polymerase (alpha-CTD). For these studies we expressed a derivative of alpha deprived of the entire C-terminal domain (alpha-Delta235) and found that expression from Pm with XylS or XylS1 was significantly decreased. To discern whether alpha-CTD activation depended on interactions with DNA and/or XylS proteins we tested a large collection of alanine substitutions within alpha-CTD. Most substitutions that had an effect on XylS and XylS1-dependent transcription were located in or adjacent to helix 1 and 4, which are known to be involved in alpha-CTD interactions with DNA. Two alanine substitutions in helix 3 (residues 287 and 291) identified a putative region of alpha-CTD/XylS regulator interactions.

Alanine↗
PubMed2001

The methionine biosynthetic pathway from homoserine in Pseudomonas putida involves the metW, metX, metZ, metH and metE gene products.

Biosynthesis of methionine from homoserine in Pseudomonas putida takes place in three steps. The first step is the acylation of homoserine to yield an acyl-L-homoserine. This reaction is catalyzed by the products of the metXW genes and is equivalent to the first step in enterobacteria, gram-positive bacteria and fungi, except that in these microorganisms the reaction is catalyzed by a single polypeptide (the product of the metA gene in Escherichia coli and the met5 gene product in Neurospora crassa). In Pseudomonas putida, as in gram-positive bacteria and certain fungi, the second and third steps are a direct sulfhydrylation that converts the O-acyl-L-homoserine into homocysteine and further methylation to yield methionine. The latter reaction can be mediated by either of the two methionine synthetases present in the cells.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed2001

A WbpL mutant of Pseudomonas putida DOT-T1E strain, which lacks the O-antigenic side chain of lipopolysaccharides, is tolerant to organic solvent shocks.

Lipopolysaccharides (LPS) are major components of the outer membrane of gram-negative bacteria and are considered a defense barrier. To determine if LPS play a role in resistance to solvents in the solvent-tolerant Pseudomonas putida DOT-T1E strain, we have generated mutants unable to synthesize the O-antigen side chain of LPS. The wbpL gene, encoding the enzyme that begins the synthesis of the O-antigen side chain of LPS of the solvent-tolerant strain, was cloned, sequenced, and knocked out in vitro with a cassette encoding kanamycin resistance, and a mutant called WbpL0 of the DOT-T1E strain was generated in vivo by site-directed mutagenesis. The WbpL mutant was compared with the wild-type strain with regard to tolerance to a number of toxic compounds, including chelating agents, organic acids, detergents, and aromatic hydrocarbons. It was found that the mutant was as tolerant as the wild-type strain to organic acids and aromatic hydrocarbons and more sensitive to ethylenediaminetetraacetic acid and deoxycholate.

Amino Acid Sequence↗
PubMed2001

Responses of Gram-negative bacteria to certain environmental stressors.

Bacteria in nature are exposed to variations in temperature, and are affected by the availability of nutrients and water and the presence of toxic molecules. Their reactions to these changes require a series of rapid adaptive responses. Although transcriptional regulation is of primary importance in these responses, translational regulation and even activation of 'silenced' enzymes are critical for survival in changing environments. Bacteria have developed a series of mechanisms at the membrane structure level to cope with high concentrations of solvents. In addition, solvent-tolerant strains express highly effective efflux pumps to remove solvents from the cytoplasm. Desiccation tolerance is based on the synthesis and accumulation of osmoprotectants together with changes in fatty acid composition to preserve membrane structure. Both cold shock and heat shock responses are mainly regulated at a post-transcriptional level, translation efficiency in the case of cold shock and mRNA half-life and sigma32 stability in the case of heat shock.

Bacterial Proteins↗
PubMed2001

Global and cognate regulators control the expression of the organic solvent efflux pumps TtgABC and TtgDEF of Pseudomonas putida.

Pseudomonas putida DOT-T1E grows on a water-toluene double liquid phase. Toluene tolerance in this microorganism is mainly achieved by at least two efflux pumps that belong to the RND family. The TtgDEF efflux pump is induced by toluene, whereas the other efflux pump, called TtgABC, is expressed at a high level in cells not exposed to toluene and at a lower level in cells grown with toluene. The ttgR gene is adjacent to the ttgABC operon and is transcribed divergently from ttgA. The expression level of ttgR was fourfold higher in cells growing in the presence of toluene than in its absence. In a TtgR-deficient background, expression from the ttgA promoter increased about 20-fold, suggesting that TtgR represses expression from the ttgA promoter. In this mutant, background expression of the ttgR gene was also much higher than in the wild-type background; however, its level of expression increased in the presence of toluene. In a ttgR mutant background, expression from the ttgD promoter followed the same pattern of expression as in the wild type. Analysis of a P. putida pTn5cat mutant that exhibited increased sensitivity to a sudden toluene shock, regardless of whether or not it was previously exposed to low toluene concentrations, revealed that pTn5cat had interrupted an lrp-like gene. The ttgR gene was expressed at very high levels in this mutant, with concomitant repression of expression of the ttgABC operon. The second ttgDEF efflux pump was expressed at low levels in this mutant strain, suggesting that the Lrp-like protein is a global regulatory protein involved in the solvent-tolerant response of this strain.

Bacterial Proteins↗
PubMed2001

Expression of a Pseudomonas putida aminotransferase involved in lysine catabolism is induced in the rhizosphere.

Using a transposon carrying a promoterless lux operon to generate transcriptional fusions by insertional mutagenesis, we have identified a Pseudomonas putida gene with increased expression in the presence of corn root exudates. Expression of the transcriptional fusion, induced by the amino acid lysine, was detected in P. putida in the rhizosphere of plants as well as in response to seed exudates. The mutant was unable to grow on lysine or delta-aminovalerate as carbon sources, which indicates that the affected function is involved in the pathway for lysine catabolism. However, the mutant strain grew with glutaric acid, the product of delta-aminovalerate metabolism via glutaric acid semialdehyde, as a C source. The translated sequence of the interrupted gene showed high levels of similarity with aminotransferases. These sets of data suggest that the product of this gene has delta-aminovalerate aminotransferase activity. This is the first direct genetic evidence correlating a DNA sequence with such activity in Pseudomonadaceae.

DNA Transposable Elements↗
PubMed2001

Dual system to reinforce biological containment of recombinant bacteria designed for rhizoremediation.

Active biological containment (ABC) systems have been designed to control at will the survival or death of a bacterial population. These systems are based on the use of a killing gene, e.g., a porin-inducing protein such as the one encoded by the Escherichia coli gef gene, and a regulatory circuit that controls expression of the killing gene in response to the presence or absence of environmental signals. An ABC system for recombinant microorganisms that degrade a model pollutant was designed on the basis of the Pseudomonas putida TOL plasmid meta-cleavage regulatory circuit. The system consists of a fusion of the Pm promoter to lacI, whose expression is controlled by XylS with 3-methylbenzoate, and a fusion of a synthetic P(lac) promoter to gef. In the presence of the model pollutant, bacterial cells survived and degraded the target compound, whereas in the absence of the aromatic carboxylic acid cell death was induced. The system had two main drawbacks: (i) the slow death of the bacterial cells in soil versus the fast killing rate in liquid cultures in laboratory assays, and (ii) the appearance of mutants, at a rate of about 10(-8) per cell and generation, that did not die after the pollutant had been exhausted. We reinforced the ABC system by including it in a Deltaasd P. putida background. A P. putida Deltaasd mutant is viable only in complex medium supplemented with diaminopimelic acid, methionine, lysine, and threonine. We constructed a P. putida Deltaasd strain, called MCR7, with a Pm::asd fusion in the host chromosome. This strain was viable in the presence of 3-methylbenzoate because synthesis of the essential metabolites was achieved through XylS-dependent induction. In the P. putida MCR7 strain, an ABC system (Pm::lacI, xylS, P(lac)::gef) was incorporated into the host chromosome to yield strain MCR8. The number of MCR8 mutants that escaped killing was below our detection limit (<10(-9) mutants per cell and generation). The MCR8 strain survived and colonized rhizosphere soil with 3-methylbenzoate at a level similar to that of the wild-type strain. However, it disappeared in less than 20 to 25 days in soils without the pollutant, whereas an asd(+), biologically contained counterpart such as P. putida CMC4 was still detectable in soils after 100 days.

Aspartic Acid↗
PubMed2001

Physiological characterization of Pseudomonas putida DOT-T1E tolerance to p-hydroxybenzoate.

Pseudomonas putida DOT-T1E was isolated as a toluene-tolerant strain. We show that it is also able to grow on high concentrations (up to 17 g/liter [123 mM]) of p-hydroxybenzoate (4HBA). Tolerance to this aromatic carboxylic acid (up to 30 g/liter [217 mM]) is improved by preexposing the cells to low 4HBA concentrations; the adaptation process is caused by the substrate itself rather than by products resulting from its metabolism. The mechanisms of 4HBA tolerance seem to involve increased rigidity of the cell membrane as a result of a decrease in the cis/trans ratio of unsaturated fatty acids. In addition, energy-dependent efflux systems seem to operate in the exclusion of 4HBA from the cell membranes.

Adaptation, Physiological↗
PubMed2001

Three efflux pumps are required to provide efficient tolerance to toluene in Pseudomonas putida DOT-T1E.

In Pseudomonas putida DOT-T1E multidrug efflux pumps of the resistance-nodulation-division family make a major contribution to solvent resistance. Two pumps have been identified: TtgABC, expressed constitutively, and TtgDEF, induced by aromatic hydrocarbons. A double mutant lacking both efflux pumps was able to survive a sudden toluene shock if and only if preinduced with small amounts of toluene supplied via the gas phase. In this article we report the identification and characterization in this strain of a third efflux pump, named TtgGHI. The ttgGHI genes form an operon that is expressed constitutively at high levels from a single promoter. In the presence of toluene the operon is expressed at an even higher level from two promoters, the constitutive one and a previously unreported one that is inducible and that partially overlaps the constitutive promoter. By site-directed mutagenesis we constructed a single ttgH mutant which was shown to be unable to survive sudden 0.3% (vol/vol) toluene shocks regardless of the preculture conditions. The mutation was transferred to single and double mutants to construct mutant strains in which two or all three pumps are knocked out. Survival analysis of induced and noninduced cells revealed that the TtgABC and TtgGHI pumps extruded toluene, styrene, m-xylene, ethylbenzene, and propylbenzene, whereas the TtgDEF pump removed only toluene and styrene. The triple mutant was hypersensitive to toluene, as shown by its inability to grow with toluene supplied via the vapor phase.

Anti-Bacterial Agents↗
PubMed2001

Mutations in genes involved in the flagellar export apparatus of the solvent-tolerant Pseudomonas putida DOT-T1E strain impair motility and lead to hypersensitivity to toluene shocks.

Pseudomonas putida DOT-T1E is a solvent-tolerant strain able to grow in the presence of 1% (vol/vol) toluene in the culture medium. Random mutagenesis with mini-Tn5-'phoA-Km allowed us to isolate a mutant strain (DOT-T1E-42) that formed blue colonies on Luria-Bertani medium supplemented with 5-bromo-4-chloro-3-indolylphosphate and that, in contrast to the wild-type strain, was unable to tolerate toluene shocks (0.3%, vol/vol). The mutant strain exhibited patterns of tolerance or sensitivity to a number of antibiotics, detergents, and chelating agents similar to those of the wild-type strain. The mutation in this strain therefore seemed to specifically affect toluene tolerance. Cloning and sequencing of the mutation revealed that the mini-Tn5-'phoA-Km was inserted within the fliP gene, which is part of the fliLMNOPQRflhBA cluster, a set of genes that encode flagellar structure components. FliP is involved in the export of flagellar proteins, and in fact, the P. putida fliP mutant was nonmotile. The finding that, after replacing the mutant allele with the wild-type one, the strain recovered the wild-type pattern of toluene tolerance and motility unequivocally assigned FliP a function in solvent resistance. An flhB knockout mutant, another gene component of the flagellar export apparatus, was also nonmotile and hypersensitive to toluene. In contrast, a nonpolar mutation at the fliL gene, which encodes a cytoplasmic membrane protein associated with the flagellar basal body, yielded a nonmotile yet toluene-resistant strain. The results are discussed regarding a possible role of the flagellar export apparatus in the transport of one or more proteins necessary for toluene tolerance in P. putida DOT-T1E to the periplasm.

Alkaline Phosphatase↗
PubMed2001

Involvement of the TonB system in tolerance to solvents and drugs in Pseudomonas putida DOT-T1E.

Pseudomonas putida DOT-T1E is able to grow with glucose as the carbon source in liquid medium with 1% (vol/vol) toluene or 17 g of (123 mM) p-hydroxybenzoate (4HBA) per liter. After random mini-Tn5'phoA-Km mutagenesis, we isolated the mutant DOT-T1E-PhoA5, which was more sensitive than the wild type to 4HBA (growth was prevented at 6 g/liter) and toluene (the mutant did not withstand sudden toluene shock). Susceptibility to toluene and 4HBA resulted from the reduced efflux of these compounds from the cell, as revealed by accumulation assays with (14)C-labeled substrates. The mutant was also more susceptible to a number of antibiotics, and its growth in iron-deficient minimal medium was inhibited in the presence of ethylenediamine-di(o-hydroxyphenylacetic acid (EDDHA). Cloning the mutation in the PhoA5 strain and sequencing the region adjacent showed that the mini-Tn5 transposor interrupted the exbD gene, which forms part of the exbBD tonB operon. Complementation by the exbBD and tonB genes cloned in pJB3-Tc restored the wild-type characteristics to the PhoA5 strain.

Alkaline Phosphatase↗
PubMed2001

Biological degradation of 2,4,6-trinitrotoluene.

Nitroaromatic compounds are xenobiotics that have found multiple applications in the synthesis of foams, pharmaceuticals, pesticides, and explosives. These compounds are toxic and recalcitrant and are degraded relatively slowly in the environment by microorganisms. 2,4,6-Trinitrotoluene (TNT) is the most widely used nitroaromatic compound. Certain strains of Pseudomonas and fungi can use TNT as a nitrogen source through the removal of nitrogen as nitrite from TNT under aerobic conditions and the further reduction of the released nitrite to ammonium, which is incorporated into carbon skeletons. Phanerochaete chrysosporium and other fungi mineralize TNT under ligninolytic conditions by converting it into reduced TNT intermediates, which are excreted to the external milieu, where they are substrates for ligninolytic enzymes. Most if not all aerobic microorganisms reduce TNT to the corresponding amino derivatives via the formation of nitroso and hydroxylamine intermediates. Condensation of the latter compounds yields highly recalcitrant azoxytetranitrotoluenes. Anaerobic microorganisms can also degrade TNT through different pathways. One pathway, found in Desulfovibrio and Clostridium, involves reduction of TNT to triaminotoluene; subsequent steps are still not known. Some Clostridium species may reduce TNT to hydroxylaminodinitrotoluenes, which are then further metabolized. Another pathway has been described in Pseudomonas sp. strain JLR11 and involves nitrite release and further reduction to ammonium, with almost 85% of the N-TNT incorporated as organic N in the cells. It was recently reported that in this strain TNT can serve as a final electron acceptor in respiratory chains and that the reduction of TNT is coupled to ATP synthesis. In this review we also discuss a number of biotechnological applications of bacteria and fungi, including slurry reactors, composting, and land farming, to remove TNT from polluted soils. These treatments have been designed to achieve mineralization or reduction of TNT and immobilization of its amino derivatives on humic material. These approaches are highly efficient in removing TNT, and increasing amounts of research into the potential usefulness of phytoremediation, rhizophytoremediation, and transgenic plants with bacterial genes for TNT removal are being done.

Aerobiosis↗