Biomedical subjects

J L Ramos

Publications and source records attributed to J L Ramos.

At least 37 records · Page 2Linked to original sources

PubMed2001

Lipoma of the liver associated with hepatocellular carcinoma and polycystic liver disease.

BACKGROUND/AIMS: We report on a patient with lipoma of the liver associated with hepatocellular carcinoma (HCC) and polycystic liver disease. METHODS: Clinical features of a patient with lipoma, HCC and polycystic liver disease are presented. A right liver lobectomy was performed. RESULTS: Histological examination revealed a poorly differentiated multicentric HCC, as well as bile duct cysts and an encapsulated tumor of adipose tissue. The postoperative course was uncomplicated. The patient died 4 years after surgery with local recurrence of HCC. CONCLUSION: We find the report to be of interest because of the unusual nature of this association and the low prevalence of lipoma of the liver. The association of liver polycystic disease with other types of tumor is discussed and the literature dealing with lipomas of the liver reviewed.

Aged↗

PubMed2000

Mutational analysis of the highly conserved C-terminal residues of the XylS protein, a member of the AraC family of transcriptional regulators.

The XylS protein of the TOL plasmid of Pseudomonas putida belongs to the so-called AraC/XylS family of regulators, that includes more than 100 different bacterial proteins. A conserved stretch of about 100 amino acids is present at the C-terminal end. This conserved region is believed to contain seven alpha-helices, including two helix-turn-helix (HTH) DNA binding motifs (alpha(2)-T-alpha(3) and alpha(5)-Talpha-(6)), connected by a linker alpha-helix (alpha(4)), and two flanking alpha-helices (alpha(1) and alpha(7)). The second HTH motif is the region with the highest homology in the proteins of the family, with certain residues showing almost 90% identity. We have constructed XylS single mutants in the most conserved residues and have analysed their ability to stimulate transcription from its cognate promoter, Pm, fused to 'lacZ. The analysis revealed that mutations in the alpha(5)-helix conserved residues had little effect on the XylS transcriptional activity, whereas the distribution of polarity in the alpha(6)-helix was important for the activity. The strongest effect of the mutations was observed in conserved residues located outside the DNA binding domain, namely, Gly-290 in the turn between the two helices, Pro-309 located downstream of alpha(6), and Leu-313, in the small last helix alpha(7), that seems to play an important role in the activation of RNA-polymerase. Our analysis shows that conservation of amino acids in the family reflects structural requirements rather than functionality in specific DNA interactions.

Amino Acid Sequence↗

PubMed2000

Degradation of o-methoxybenzoate by a two-member consortium made up of a gram-positive Arthrobacter strain and a gram-negative Pantotea strain.

Aromatic carboxylic acids substituted with methoxylated groups are among the most abundant products in "alpechin", the wastes resulting from pressing olives to obtain olive oil. Degradation of o-methoxybenzoate by an stable consortium made of a gram positive bacterium, Arthrobacter oxydans, and gram negative one, Pantotea agglomerans, was shown to mineralize this compound efficiently. The concerted action of both microorganisms was needed for the two first steps in the process, namely, the conversion of o-methoxybenzoate into salycilate, and the hydroxylation of the latter to gentisate. Gentisate was further degraded by the Arthrobacter strain.

Anisoles↗

PubMed2000

Retrotransfer of DNA in the rhizosphere.

Retrotransfer of DNA refers to the phenomenon by which a plasmid travels from a host strain to a recipient one and returns to the original host, bringing with it DNA from the recipient. The resultant host strain with DNA from the recipient is called a retrotransconjugant. The retrotransfer phenomenon mediated by the TOL plasmid pWW0 and other plasmids has been documented on plates under optimal laboratory culture conditions, but never under natural conditions. In this work, we show that retrotransfer mediated by the IncP9 TOL pWW0 plasmid occurs in the rhizosphere, a niche in which the continuous supply of nutrients via root exudates allows cells to reach a high density. This suggests that this unusual sexual fertilization may be of great importance in lateral gene transfer. We also show that retrotransfer of DNA seems to require co-integration of the plasmid and the host chromosome and subsequent resolution, because a TOL plasmid with a mutation in the tnpR gene, encoding the resolvase of the Tn4653 of the TOL plasmid, was self-transferred between Pseudomonas strains, but unable to mobilize chromosome.

Blotting, Southern↗

PubMed2000

Control of expression of divergent Pseudomonas putida put promoters for proline catabolism.

Pseudomonas putida KT2440 uses proline as the sole C and N source. Utilization of this amino acid involves its uptake, which is mediated by the PutP protein, and its conversion into glutamate, mediated by the PutA protein. Sequence analysis revealed that the putA and putP genes are transcribed divergently. Expression from the putP and putA genes was analyzed at the mRNA level in different host backgrounds in the absence and presence of proline. Expression from the put promoters was induced by proline. The transcription initiation points of the putP and putA genes were precisely mapped via primer extension, and sequence analysis of the upstream DNA region showed well-separated promoters for these two genes. The PutA protein acts as a repressor of put gene expression in P. putida because expression from the put promoters is constitutive in a host background with a knockout putA gene. This regulatory activity is independent of the catabolic activity of PutA, because we show that a point mutation (Glu896-->Lys) that prevents catalytic activity allowed the protein to retain its regulatory activity. Expression from the put promoters in the presence of proline in a putA-proficient background requires a positive regulatory protein, still unidentified, whose expression seems to be sigma(54) dependent because the put genes were not expressed in a sigma(54)-deficient background. Expression of the putA and putP genes was equally high in the presence of proline in sigma(38)- and ihf-deficient P. putida backgrounds.

Amino Acid Transport Systems, Neutral↗

PubMed2000

Proline catabolism by Pseudomonas putida: cloning, characterization, and expression of the put genes in the presence of root exudates.

Pseudomonas putida KT2442 is a root-colonizing strain which can use proline, one of the major components in root exudates, as its sole carbon and nitrogen source. A P. putida mutant unable to grow with proline as the sole carbon and nitrogen source was isolated after random mini-Tn5-Km mutagenesis. The mini-Tn5 insertion was located at the putA gene, which is adjacent to and divergent from the putP gene. The putA gene codes for a protein of 1,315 amino acid residues which is homologous to the PutA protein of Escherichia coli, Salmonella enterica serovar Typhimurium, Rhodobacter capsulatus, and several Rhizobium strains. The central part of P. putida PutA showed homology to the proline dehydrogenase of Saccharomyces cerevisiae and Drosophila melanogaster, whereas the C-terminal end was homologous to the pyrroline-5-carboxylate dehydrogenase of S. cerevisiae and a number of aldehyde dehydrogenases. This suggests that in P. putida, both enzymatic steps for proline conversion to glutamic acid are catalyzed by a single polypeptide. The putP gene was homologous to the putP genes of several prokaryotic microorganisms, and its gene product is an integral inner-membrane protein involved in the uptake of proline. The expression of both genes was induced by proline added in the culture medium and was regulated by PutA. In a P. putida putA-deficient background, expression of both putA and putP genes was maximal and proline independent. Corn root exudates collected during 7 days also strongly induced the P. putida put genes, as determined by using fusions of the put promoters to 'lacZ. The induction ratio for the putA promoter (about 20-fold) was 6-fold higher than the induction ratio for the putP promoter.

Amino Acid Sequence↗

PubMed2000

Mutations in each of the tol genes of Pseudomonas putida reveal that they are critical for maintenance of outer membrane stability.

The outer membrane of gram-negative bacteria functions as a permeability barrier that protects cells against a large number of antibacterial agents. OprL protein of Pseudomonas putida has been shown to be crucial to maintain the stability of this cell component (J. J. Rodríguez-Herva, M.-I. Ramos-González, and J. L. Ramos. J. Bacteriol. 178:1699-1706, 1996). In the present study we cloned and mutagenized the orf1, tolQ, tolR, tolA, and tolB genes from P. putida KT2440, which were located upstream of the oprL gene. Polar and nonpolar mutations of the P. putida tolQ, tolR, tolA, and tolB genes were generated in vitro by using the omega-Km(r) interposon, which carries two transcriptional stop signals, or a promoterless xylE cassette, lacking any transcriptional stop signal, respectively. The mutant constructs were used to inactivate, by reverse genetics procedures, the corresponding chromosomal copies of the genes. The phenotype of each mutant strain was analyzed and compared with those of the wild-type strain and the previously characterized P. putida oprL::xylE mutant. All mutant strains exhibited a similar phenotype: altered cell morphology, bleb formation at the cell surface, release of periplasmic and outer membrane proteins to the extracellular medium, increased sensitivity to a variety of compounds (i.e., EDTA, sodium dodecyl sulfate, deoxycholate, and some antibiotics), filament formation, and severely reduced cell motility. Altogether, these results demonstrate the importance of the Tol-OprL system for the maintenance of outer membrane integrity in P. putida and suggest a possible role of these proteins in assembling outer membrane components.

Bacterial Outer Membrane Proteins↗

PubMed2000

Transcription activation by a variety of AraC/XylS family activators does not depend on the class II-specific activation determinant in the N-terminal domain of the RNA polymerase alpha subunit.

The N-terminal domain of the RNA polymerase alpha subunit (alpha-NTD) was tested for a role in transcription activation by a variety of AraC/XylS family members. Based on substitutions at residues 162 to 165 and an extensive genetic screen we conclude that alpha-NTD is not an activation target for these activators.

Bacterial Proteins↗

PubMed2000

A set of genes encoding a second toluene efflux system in Pseudomonas putida DOT-T1E is linked to the tod genes for toluene metabolism.

Sequence analysis in Pseudomonas putida DOT-T1E revealed a second toluene efflux system for toluene metabolism encoded by the ttgDEF genes, which are adjacent to the tod genes. The ttgDEF genes were expressed in response to the presence of aromatic hydrocarbons such as toluene and styrene in the culture medium. To characterize the contribution of the TtgDEF system to toluene tolerance in P. putida, site-directed mutagenesis was used to knock out the gene in the wild-type DOT-T1E strain and in a mutant derivative, DOT-T1E-18. This mutant carried a Tn5 insertion in the ttgABC gene cluster, which encodes a toluene efflux pump that is synthesized constitutively. For site-directed mutagenesis, a cassette to knock out the ttgD gene and encoding resistance to tellurite was constructed in vitro and transferred to the corresponding host chromosome via the suicide plasmid pKNG101. Successful replacement of the wild-type sequences with the mutant cassette was confirmed by Southern hybridization. A single ttgD mutant, DOT-T1E-1, and a double mutant with knock outs in the ttgD and ttgA genes, DOT-T1E-82, were obtained and characterized for toluene tolerance. This was assayed by the sudden addition of toluene (0.3% [vol/vol]) to the liquid culture medium of cells growing on Luria-Bertani (LB) medium (noninduced) or on LB medium with toluene supplied via the gas phase (induced). Induced cells of the single ttgD mutant were more sensitive to sudden toluene shock than were the wild-type cells; however, noninduced wild-type and ttgD mutant cells were equally tolerant to toluene shock. Noninduced cells of the double DOT-T1E-82 mutant did not survive upon sudden toluene shock; however, they still remained viable upon sudden toluene shock if they had been previously induced. These results are discussed in the context of the use of multiple efflux pumps involved in solvent tolerance in P. putida DOT-T1E.

Amino Acid Sequence↗

PubMed2000

Respiration of 2,4,6-trinitrotoluene by Pseudomonas sp. strain JLR11.

Under anoxic conditions Pseudomonas sp. strain JLR11 can use 2,4, 6-trinitrotoluene (TNT) as the sole N source, releasing nitrite from the aromatic ring and subsequently reducing it to ammonium and incorporating it into C skeletons. This study shows that TNT can also be used as a terminal electron acceptor in respiratory chains under anoxic conditions by Pseudomonas sp. strain JLR11. TNT-dependent proton translocation coupled to the reduction of TNT to aminonitrotoluenes has been observed in TNT-grown cells. This extrusion did not occur in nitrate-grown cells or in anaerobic TNT-grown cells treated with cyanide, a respiratory chain inhibitor. We have shown that in a membrane fraction prepared from Pseudomonas sp. strain JLR11 grown on TNT under anaerobic conditions, the synthesis of ATP was coupled to the oxidation of molecular hydrogen and to the reduction of TNT. This phosphorylation was uncoupled by gramicidin. Respiration by Pseudomonas sp. strain JLR11 is potentially useful for the biotreatment of TNT in polluted waters and soils, particularly in phytorhizoremediation, in which bacterial cells are transported to the deepest root zones, which are poor in oxygen.

Adenosine Triphosphate↗

PubMed2000

Genetic analysis of functions involved in adhesion of Pseudomonas putida to seeds.

Many agricultural uses of bacteria require the establishment of efficient bacterial populations in the rhizosphere, for which colonization of plant seeds often constitutes a critical first step. Pseudomonas putida KT2440 is a strain that colonizes the rhizosphere of a number of agronomically important plants at high population densities. To identify the functions involved in initial seed colonization by P. putida KT2440, we subjected this strain to transposon mutagenesis and screened for mutants defective in attachment to corn seeds. Eight different mutants were isolated and characterized. While all of them showed reduced attachment to seeds, only two had strong defects in their adhesion to abiotic surfaces (glass and different plastics). Sequences of the loci affected in all eight mutants were obtained. None of the isolated genes had previously been described in P. putida, although four of them showed clear similarities with genes of known functions in other organisms. They corresponded to putative surface and membrane proteins, including a calcium-binding protein, a hemolysin, a peptide transporter, and a potential multidrug efflux pump. One other showed limited similarities with surface proteins, while the remaining three presented no obvious similarities with known genes, indicating that this study has disclosed novel functions.

Bacterial Adhesion↗

PubMed2000

[Pyogenic and non-pyogenic vertebral osteomyelitis: descriptive and comparative study of a series of 40 cases].

BACKGROUND: To describe the features of vertebral osteomyelitis (VO) in our area, and to compare pyogenic VO (PVO) and non-pyogenic VO (NPVO). METHODS: Retrospective study of patients with VO diagnosed in our hospital between january 1992 and december 1998. RESULTS: We identify 40 patients with VO: 17 (42.5%) with tuberculous VO, 12 (30%) with PVO, 10 (25%) with brucellar VO and 1 (2.5%) with a VO caused by Candida albicans. The mean age was 52.9 years, and 75% of patients were male. Only a 20% of cases had a known immunosuppression. Fever/febricule was present in 55% of patients and vertebral pain in 95%. The more frequent locations were lumbar (67.5%) and dorsal (27.5%). One or more paravertebral or epidural abscesses were present in 67.5% of cases and spinal cord compression was present in 27.5%. An elevated erythrocyte sedimentation rate (ESR) was present in 40% and leukocytosis in 27.5%. Sequelae after complete treatment occurred in 40% of patients, with residual vertebral pain in 37.5%. There was a shorter clinical course and a higher frequency of fever/febricule and leukocytosis in PVO than in NPVO. CONCLUSIONS: In our area the more frequent etiology of VO is tuberculosis. VO occurs predominantly in males older than 50 years, without known immunosuppression, and almost exclusively with lumbar or dorsal locations. The absence of fever/febricule, leukocytosis or elevated ESR do not exclude the diagnosis of VO. Sequelae after complete treatment are relatively frequent, fundamentally residual vertebral pain. There are some differences in the features between PVO and NPVO, which may aid in the differential diagnosis of both entities.

Adolescent↗

PubMed1999

Toluene metabolism by the solvent-tolerant Pseudomonas putida DOT-T1 strain, and its role in solvent impermeabilization.

Pseudomonas putida DOT-T1E is a solvent-tolerant strain able to grow with toluene as the sole C-source. Tn5 mutagenesis was carried out and a mutant unable to use toluene as the sole C-source was isolated. DNA was sequenced upstream and downstream of the site where the Tn5 was inserted. Analysis of the DNA revealed 13 open reading frames (ORFs) homologous to the tod genes for the toluene dioxygenase pathway of P. putida F1, which are organized in two operons: todXFC1C2BADEGIH and todST. The Tn5 was inserted at the todH gene. The role of the todXFC1C2BADEGIH operon in toluene metabolism was further confirmed in a todC1 mutant (generated by insertional inactivation), which was unable to use toluene as the sole C-source. Primer extension analysis identified a single promoter upstream from the todX gene. The -10 and -35 regions of this promoter showed no significant homology to known promoters. Expression from the todX promoter occurred in response to toluene, ethylbenzene, styrene, xylenes and other aromatic hydrocarbons. Expression from the todS gene was constitutive. Sensitivity to toluene of the todH and todC1 mutants was similar to that of the wild-type strain. This suggests that toluene metabolism is not involved in toluene tolerance.

Base Sequence↗

PubMed1999

Critical nucleotides in the upstream region of the XylS-dependent TOL meta-cleavage pathway operon promoter as deduced from analysis of mutants.

The Pm promoter, dependent on TOL plasmid XylS regulator, which is activated by benzoate effectors, drives transcription of the meta-cleavage pathway for the metabolism of alkylbenzoates. This promoter is unique in that in vivo transcription is mediated by RNA-polymerase with different sigma factors. In vivo footprinting analysis shows that XylS interacted with nucleotides in the -40 to -70 region. In vivo and in vitro methylation of Pm shows extensive methylation of T at position -42 in the bottom strand, suggesting that it represents a key distortion point that may favor XylS/RNA polymerase interactions. Methylation of T-42 was highest in cells bearing XylS and in the presence of an effector. Gs in the -47 to -61 region appeared to be more protected in cells harboring XylS in the presence than in the absence of the effector. Almost 100 mutants in the Pm region between -41 and -78 were generated; transcriptional analysis of these mutants defined the XylS target as two direct repeats with the sequence TGCAN6GGNCA. These motifs cover the -70 to -56 and the -49 to -35 regions. Single point mutations revealed that nucleotides located at -49 to -46 and at -59, -60, -62, and -70 are the most critical for appropriate XylS-Pm interactions.

Bacterial Proteins↗

PubMed1999

Is glycosuria a reliable indicator of adequacy of glucose infusion rate in preterm infants?

CONTEXT: Adequacy of glucose infusion may be monitored via the glycosuria levels, as there is a relationship between glycemia and glycosuria regulated by the renal glucose threshold. In the neonatal period, however, this relationship is not so clear. OBJECTIVE: To evaluate the occurrence of glycosuria in preferm infants submitted to glucose infusion and to verify the relationship between glycosuria and blood glucose level. DESIGN: Accuracy study. SETTING: Neonatal intensive care unit of General Maternity Hospital. PATIENTS: 40 Preterm newborns receiving glucose infusion. PROCEDURES: 511 concomitant determinations of glycemia and glycosuria were performed. These 511 pairs were divided into stable and unstable, according to the clinical status of the newborn at the time of data collection, and they were studied in relation to the gestational age, birth weight and glucose infusion rate. RESULTS: The results revealed a greater frequency of glycosuria in gestational age < or = 30 weeks, birth weight < 1500 g and glucose infusion rate > 6 mg/kg/min. Eight (25.8%) episodes of positive glycosuria occurred in the absence of hyperglycemia, indicating only a moderate concordance between them. CONCLUSION: Glycosuria alone is an unreliable marker of blood glucose concentration and adequacy of glucose infusion rate. It is therefore necessary to monitor blood glucose levels in infants submitted to continuous glucose infusion.

Biomarkers↗

PubMed1999

A risk factor for early-onset infection in premature newborns: invasion of chorioamniotic tissues by leukocytes.

The authors report a prospective study of correlation between histopathological alterations of the placenta, risk factors and early-onset bacterial infections in 224 premature newborns. They used a mathematical model for evaluation and prediction of neonatal bacterial infection according to the localization in chorioamniotic tissues (chorioamniotic plate, amniotic membranes and umbilical cord) invaded by leukocytes. Septicemia, pneumonia or omphalitis were documented in 45 (20%) infected premature newborns and inflammatory lesions in the placenta were observed in all of them. In order of statistical significance, the most important variables for early-onset bacterial neonatal infection were invasion of the chorioamniotic plate, amniotic membranes and umbilical cord tissues by PMNL (P < 0.0000), premature rupture of membranes (P < 0.0000), birthweight lower than 1500 g (P < 0.0000), gestational age under 34 weeks (P < 0.0001), foul smell (P < 0.0038), no antibiotics before delivery (P < 0.0066) and intrapartum fever (P < 0.0087). By logistic stepwise multiple regression analysis, invasion of fetal chorioamniotic plate and of amniotic membranes by leukocytes were the only statistically significant variables. The probability of neonatal infection in premature newborns, when polymorphonuclear neutrophils were present in chorioamniotic plate and in amniotic membranes, was 62.5%, while the probability was 0.5% when these tissues were normal. These data suggest that histological chorioamnionitis has to be considered as an important risk factor for early-onset infection in premature newborns.

Age of Onset↗

PubMed1999

Prediction of length of hospital stay in neonatal units for very low birth weight infants.

OBJECTIVE: To develop models for estimating the length of hospital stay (LOS) of very low birth weight infants (VLBW), based on perinatal risk factors present during the first week of life and during the entire hospitalization period. STUDY DESIGN: The files of 155 VLBW were analyzed, and the influence of individual risk factors were initially evaluated by univariate analysis, using multiple-regression. Two mathematical models were built to estimate the LOS. RESULTS: The first model, using risk factors present during the first 3 days of life, is as follows: LOS = -0.074A + 22.06B + 22.85C - 16.78D - 2.07E + 10.51F + 203.12 (R2 = 0.63). (The letters are added to show what each number represents: A: birth weight; B: occurrence of respiratory distress syndrome; C: endotracheal intubation during resuscitation; D: 1-minute Apgar score; E: gestational age; F: presence of complications during delivery.) The second model, using factors present during the entire hospitalization period, is: LOS = 0.61G + 29.19H + 24.68I + 14.21J + 23.56K + 9.54L + 7.41M + 20.43 (R2 = 0.82). (G: age receiving nutritional support of > or = 120 kcal/kg per day; H: occurrence of systemic candidiasis; I: birth weight < 1000 gm; J: presence of delivery complication; K: occurrence of bronchopulmonary dysplasia; L: birth weight > or = 1000 gm and < or = 1249 gm; M: occurrence of anemia). CONCLUSION: Both models are applicable for estimating the hospitalization period, and the addition of variables present during the entire hospitalization period improved the accuracy of the model.

Brazil↗

PubMed1999

The XylS-dependent Pm promoter is transcribed in vivo by RNA polymerase with sigma 32 or sigma 38 depending on the growth phase.

The Pm promoter of the TOL plasmid of Pseudomonas putida is expressed at high level along the growth curve. This transcription is dependent on the positive regulator XylS activated by 3-methylbenzoate. The sigma factor sigma 38 is required for expression in early stationary phase and thereafter. To test whether sigma 70 was involved in Pm transcription in exponential phase, we have followed mRNA synthesis in a rpoD thermosensitive strain. No difference in Pm transcription was found between the wild type and the thermosensitive strain at the restrictive temperature of 42 degrees C, indicating that transcription was independent of the sigma factor sigma 70. However, basal levels of mRNA expression from Pm in this strain in exponential phase were more than twofold higher at 42 degrees C, suggesting involvement of sigma 32 in Pm transcription. In a rpoH background, no expression of Pm took place in the exponential phase, whereas it increased during stationary phase, and in a rpoH rpoS double mutant no activity from the Pm promoter was detected along the growth curve. We have shown that the increase in the amount of sigma 32 factor necessary for transcription in exponential phase is provided through induction of the heat shock response by the presence of the effector 3-methylbenzoate, which is also required for activation of the positive regulator XylS. We conclude that activation of Pm transcription is achieved through a switch between two stress-responsive factors, sigma 32 in exponential phase and sigma 38 in stationary phase. In both cases, transcription is dependent on the activator XylS and presents the same transcription start point.

Bacterial Proteins↗