Biomedical subjects

E Carvajal

Publications and source records attributed to E Carvajal.

At least 19 recordsLinked to original sources

PubMed2001

Herpesvirus-6 encephalitis complicated by Wernicke-Korsakoff syndrome in a pediatric recipient of unrelated cord blood transplantation.

A 10-year-old girl with M2 acute myeloid leukemia underwent an unrelated cord blood transplantation in refractory first relapse. On day +13, after 48 hours with fever, she showed a measles-like rash, and on day +15, she began experiencing neurologic symptoms (headache, tremors, weakness, nystagmus, mild confusion, speaking, taste, and behavior disturbances, and focal seizures). She also had amnesia for recent events with disability to learn, mimicking Wernicke-Korsakoff syndrome. Computed tomography of the brain and cerebrospinal fluid (CSF) and electroencephalogram were nonspecific. We found human herpesvirus 6 (HHV-6) DNA in CSF and cytomegalovirus in bronchoalveolar lavage using polymerase chain reaction techniques. Treatment with ganciclovir and foscarnet was effective, with total resolution of symptoms.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2001

The pleitropic drug ABC transporters from Saccharomyces cerevisiae.

The Saccharomyces cerevisiae genome contains 16 genes encoding full-size ABC transporters. Each comprises two nucleotide binding folds (NBF) alternating with transmembrane domains (TM). We have studied in detail three plasma membrane multidrug exporters: Pdr5p (TC3.A.1.205.1) and Snq2p (TC3.A.1.205.2) which share NBF-TM-NBF-TM topology as well as Yor1p (TC3.A.1.208.3) which exhibits the reciprocal TM-NBF-TM-NBF topology. The substrate specificity of Pdr5p, Snq2p and Yor1p are largely, but not totally, overlapping as shown by screening the growth inhibition by 349 toxic compounds of combinatorial deletants of these three ABC genes. Multiple deletion of 7 ABC genes (YOR1, SNQ2, PDR5, YCF1, PDR10, PDR11 and PDR15) and of two transcription activation factors (PDR1 and PDR3) renders the cell from 2 to 200 times more sensitive to numerous toxic coumpounds including antifungals used in agriculture or medicine. The use of the pdr1-3 activating mutation and when necessary of the PDR5 promoter in appropriate multideleted hosts allow high levels of expression of Pdr5p, Snq2p or Yor1 p. These overexpressed proteins exhibit ATPase activity in vitro and confer considerable multiple drug resistance in vivo. The latter property can be used for screening specific inhibitors of fungal and other ABC transporters.

ATP-Binding Cassette Transporters↗

PubMed2000

Nitrogen regulation of Saccharomyces cerevisiae invertase. Role of the URE2 gene.

The regulation of extracellular enzymes is of great biotechnological interest. We studied the regulatory role of the URE2 gene on the periplasmic invertase of Saccharomyces cerevisiae, because its periplasmic asparaginase is regulated by the URE2/GLN3 system. Enzymatic activity was measured in the isogenic strains P40-1B, the ure2 mutant P40-3C, and the P40-3C strain transformed with the pIC-CS plasmid carrying the URE2 gene. The assays were performed using midlog and stationary phase cells and nitrogen-starved cells from these growth phases. During exponential growth, the level of invertase in both wild-type and ure2 mutant cells was comparable. However, the invertase activity in ure2 mutant cells from stationary phase was sixfold lower than in the wild-type cells. When P40-3C cells were transformed with the pIC-CS plasmid, the wild-type phenotype was restored. On nitrogen starvation in the presence of sucrose, the invertase activity in wild-type cells from midlog phase decreased three times, whereas in stationary cells, the activity decreased eight times. However, invertase activity doubled in ure2 mutant cells from both phases. When these cells were transformed with the aforementioned plasmid, the wild-type phenotype was restored, although a significant invertase decrease in stationary cell was not observed. These results suggested that the URE2 protein plays a role in invertase activity.

Asparaginase↗

PubMed1999

L-asparaginase II of saccharomyces cerevisiae. Activity profile during growth using an ure2 mutant P40-3C and a P40-3C + URE2p strain.

The activity profile of the periplasmic asparaginase of Saccharomyces cerevisiae was determined during cell growth in an ure2 mutant; in an ure2 transformed with a plasmid containing the gene URE2 and, for comparison, in the strain D273-10B. Cells were cultivated in media presenting variable quantitative and qualitative nitrogen availability and the enzyme activity was evaluated in fresh and in nitrogen-starved cells. Nitrogen affected the asparaginase II level in fresh and starved cells of all strains. In the best condition, enzyme was produced by the wild-type cells at the late log-phase in the glucose/ammonium medium with a carbon to nitrogen ratio 4.3:1. Upon starvation, the activity doubled. The overall profile of the transformed strain was similar to that of the wild-type strain. In the ure2 mutant, high-enzyme levels were observed during growth, as expected. However the activity level, upon starvation, in proline grown cells, increased sixfold, suggesting that in addition to the Ure2p-Gln3p system, another system regulates asparaginase II biosynthesis.

Asparaginase↗

PubMed1997

Asparaginase II of Saccharomyces cerevisiae. GLN3/URE2 regulation of a periplasmic enzyme.

The production of some extracellular enzymes is known to be negatively affected by readily metabolized nitrogen sources such as NH4+ although there is no consensus regarding the involved mechanisms. Asparaginase II is a periplasmic enzyme of Saccharomyces cerevisiae encoded by the ASP3 gene. The enzyme activity is not found in cells grown in either ammonia, glutamine, or glutamate, but it is found in cells that have been subjected to nitrogen starvation or have been grown on a poor source of nitrogen such as proline. In this report it is shown that the formation of this enzyme is dependent upon the functional GLN3 gene and that the response to nitrogen availability is under the control of the URE2 gene product. In this respect the expression of ASP3 is similar to the system that regulates the GLN1, GDH2, GAP1, and PUT4 genes that codes for glutamine synthetase, NAD-linked glutamate dehydrogenase, general amino-acid permease, and high affinity proline permease, respectively.

Asparaginase↗

PubMed1997

Molecular and phenotypic characterization of yeast PDR1 mutants that show hyperactive transcription of various ABC multidrug transporter genes.

Mutations at the yeast PDR1 transcriptional regulator locus are responsible for overexpression of the three ABC transporter genes PDR5, SNQ2 and YOR1, associated with the appearance of multiple drug resistance. The nucleotide sequences of 13 alleles of PDR1, comprising 6 multidrug resistance mutants, 1 intragenic suppressor and 6 wild types, have been determined. Single amino acid substitutions were shown to result from the mutations pdr1-2 (M308I), pdr1-3 (F815S), pdr1-6 (K302Q), pdr1-7 (P298A) and pdr1-8 (L1036 W), whereas the intragenic suppressor mutant pdr1-100 is deleted for the two amino acids L537 and A538. An isogenic series of strains was constructed containing the mutant alleles pdr1-3, pdr1-6 and pdr1-8 integrated into the genome. We found that the levels of resistance to cycloheximide, oligomycin, 4-nitroquinoline-N-oxide and ketoconazole were increased in all three mutants. The increase was more pronounced in the pdr1-3 than in the pdr1-6 and pdr1-8 mutants. Studies of the activity of the promoters of the ABC genes PDR5, SNQ2 and YOR1 demonstrated that the combination of the PDR5 promoter and the pdr1-3 mutation resulted in the highest level of promoter induction. Concomitantly, the level of PDR5 mRNA, of Pdr5p protein, and of its associated nucleoside triphosphatase activity, was strongly increased in the plasma membranes of the PDR1 mutants. Again, the pdr1-3 allele was associated with a stronger effect than the pdr1-8 and pdr1-6 alleles. The locations of the mutations in the PDR1 gene indicate that at least three different regions distributed throughout the Pdr1p transcription factor may be mutated to generate a Pdr1p with considerably increased transcriptional activation potency. These gain-of-function mutations support the concept, recently proposed, that in members of the large family of yeast Zn2Cys6 transcription factors a central inhibitory domain exists (delineated by the pdr1-7, pdr1-6 and pdr1-2 mutations). This domain may interact in a locked conformation with a putative, more C-terminally located inhibitory domain (mutated in pdr1-3), and with the putative activation domain (mutated in pdr1-8).

ATP-Binding Cassette Transporters↗

PubMed1996

Assay for in vivo yeast invertase activity using NaF.

The methods used for invertase activity determination are based on the measurement of glucose or reducing sugars produced by the enzymatic hydrolysis of sucrose into glucose and fructose. When whole yeast cells are used in these assays, the monosaccharides formed by the action of the periplasmic enzyme can be taken up and metabolized, leading to errors on the enzyme activity determination. This study reports a method for a more accurate invertase activity measurement by blocking the glycolytic pathway. In this method the cells were preincubated with 50 mM sodium fluoride, and inhibitor of enolase. This in vivo measurement of the enzyme activity, under initial rate conditions, was performed using cell concentrations up to 64 mg cell/ml. The results obtained showed that this method is particularly useful for cells with low invertase activity.

Enzyme Inhibitors↗

PubMed1995

Analysis of second-site mutations that suppress the multiple drug resistance phenotype of the yeast PDR1-7 allele.

The yeast PDR1 locus encodes a member of the C6 zinc cluster family of transcriptional regulatory proteins. Among the targets of PDR1 is the yeast PDR5 locus. The product of this gene is a member of the ATP-binding cassette (ABC) transmembrane protein family and plays a major role in inhibitor efflux. Mutations in PDR1 affect the relative level of PDR5 transcript and can therefore result in increased or decreased drug resistance. We isolated three second-site suppressors of a PDR1-7 semidominant hyper-resistant mutation. These mutants were drug hypersensitive, as compared with isogenic controls. Two of the three mutations contained alterations in a putative DNA-binding domain. Significantly, the mutant proteins exhibited reduced DNA-binding capacity.

ATP-Binding Cassette Transporters↗

PubMed1994

PDR3, a new yeast regulatory gene, is homologous to PDR1 and controls the multidrug resistance phenomenon.

The Saccharomyces cerevisiae PDR3 gene, located near the centromere of chromosome II, has been completely sequenced and characterised. Mutations pdr3-1 and pdr3-2, which confer resistance to several antibiotics can be complemented by a wild-type allele of the PDR3 gene. The sequence of the wild-type PDR3 gene revealed the presence of a long open reading frame capable of encoding a 976-amino acid protein. The protein contains a single Zn(II)2Cys6 binuclear-type zinc finger homologous to the DNA-binding motifs of other transcriptional activators from lower eukaryotes. Evidence that the PDR3 protein is a transcriptional activator was provided by demonstrating that DNA-bound LexA-PDR3 fusion proteins stimulate expression of a nearby promoter containing LexA binding sites. The use of LexA-PDR3 fusions revealed that the protein contains two activation domains, one localised near the N-terminal, cysteine-rich domain and the other localised at the C-terminus. The salient feature of the PDR3 protein is its similarity to the protein coded by PDR1, a gene responsible for pleiotropic drug resistance. The two proteins show 36% amino acid identity over their entire length and their zinc finger DNA-binding domains are highly conserved. The fact that the absence of both PDR1 and PDR3 (simultaneous disruption of the two genes) enhances multidrug sensitivity strongly suggests that the two transcriptional factors have closely related functions.

Amino Acid Sequence↗

PubMed1994

Economic study of neutropenia induced by myelotoxic chemotherapy.

This article describes the economic and social impact of neutropenia induced by myelotoxic chemotherapy in patients with cancer during the period 1 January-31 December 1991. Neutropenia is a life-threatening complication of chemotherapy in patients with cancer. The episodes of fever and infections originating from neutropenia require hospitalization of the patient until the granulocyte levels are restored. The calculation of the economic cost was based on the following parameters: length of stay in hospital, analytical tests performed on the patient, type and cost of drug therapy administered, blood transfusions performed, health assistance received, cost of isolation and absence from work. The overall economic cost of neutropenia in patients with cancer reached 329,775 pesetas ($2,893). Cost of the health-care staff was the largest budget item in relation to the total health resources estimated.

Adolescent↗

PubMed1994

[Pollen of gramineae and its relation with allergic manifestations in Costa Rica].

Allergy to pollen is a significant health problem in Costa Rica. This volume both reviews and presents new data on the subject, for which there are no similar comprehensive studies in other areas of Latin America. A description of the sampling methods is presented, as well as an introduction to work presented throughout the volume. A hundred control subjects were compared with 480 patients with rhinitis, conjuntivitis, bronchial asthma and/or atopic dermatitis. Poaceae pollen ranks first in Type I allergic sensitivization, because (1) it produces abundant pollen, (2) occurs in a variety of climates and habitats and (3) its proteins are particularly allergenic. In 1986, about 0.33% of the country's population became new allergy patients, and the rate is increasing. The normal extraction methods were satisfactory and were applied to frozen pollen with a mean active life of about three years. Prich tests were done for a period of two years using only extract combinations by tribe. A second stage used species-specific extracts (1/10 W/V 50% glycerine).

Allergens↗

PubMed1994

[Type I hypersensitivity to graminea pollens by tribe, in allergic rhinitis patients].

Type I Hypersensitivity to pollens of the Poaceae Family (Gramineae) organized by subfamilies and tribes were studied in 404 Costa Rican patients with a clinical diagnosis of Allergic Rhinitis and 100 non atopic subjects. Skin prick tests (for each tribe) were done with pooled samples from the species which are most abundant in Costa Rica (1/10 W/V, 50% glycerinated). The tribes were: Paniceae, Andropogoneae, Poeae, Aveneae, Oryzoideae and Chloridoideae (Eragrostoideae); 53% of the patients were positive to at least one of the tribes against 2% of the non atopic subjects. The most frequent tribe was the Aveneae with a 37.4% of positivity; the lowest was Paniceae with an 24.8% positivity. The provinces with the highest positivities to graminean pollens were Alajuela and Guanacaste with more than 60% of the rhinitic patients positive for at least one test. When an individual is positive to one tribe, it does not mean that this patient is positive to other tribes of this large Family. It is necessary to include all the tribes of the Poaceae Family present in a country or region to properly analyze allergic hypersensitivity to this family.

Adolescent↗

PubMed1994

[Type I hypersensitivity to pollen of the Poaceae family (Gramineae), by tribe, in atopic dermatitis patients].

Type I Hypersensitivity to Poaceae pollen was tested in 27 Costa Rican patients with a clinical diagnosis of Atopic Dermatitis (six were males). Skin prick tests were done with 1/10 W/V, 50% glycerinated, pollen extracts, organized by tribes. Most patients had several allergic problems. Sensitivity increased with the number of allergic diagnoses. Positivity ranged from 25% in patients with Atopic Dermatitis exclusively to 100% in those with three or four simultaneous allergic illnesses. Andropogoneae was highest with 37% while the lower positivity was in the Paniceae (22.2%).

Adolescent↗

PubMed1994

[Type I hypersensitivity to gramineae pollen (by tribe), in allergic conjunctivitis patients].

A total of 84 Costa Rican patients with a clinical diagnosis of Allergic Conjuntivitis (with or without other allergic diagnosis) and 100 non atopic subjects were studied by prick tests for all of Poaceae Tribes present in Costa Rica. Positivity was recorded in 70% of the Allergic Conjunctivitis patients for at least one of the tribes (against 2% of controls). An increase in the number of analysed tribes correlated with a higher number of patients allergic to graminean pollens. Guanacaste Province had the highest positivity, and patients aged 11 to 20 years were the most sensitive. Aveneae had the highest positivity except in Alajuela Province, in which Paniceae was highest in positivity. Poaceae pollens are very important in allergic hypersensitivity among Allergic Conjunctivitis patients. Prick tests for all the tribes present in the country are required to fully detect positivity to this family.

Adolescent↗

PubMed1994

[Type I hypersensitivity to gramineae pollen (by tribe), in bronchial asthma patients].

A total of 260 Costa Rican patients (106 males) with clinical diagnosis of Bronchial Asthma and 100 non atopic subjects were studied with skin prick tests for all Poaceae tribes. Allergic Type I Hypersensitivity to these pollens among the Bronchial Asthma patients reached 51.2% positivity to at least one tribe (2% in controls). The positivity to each tribe was lower than positivity to at least one tribe. Up to 25.4% of positive patients could missed by not testing for all tribes present in the country. The pollen of the Oryzeae tribe (rice) showed the highest positivity among the Bronchial Asthma patients of Guanacaste Province. This province produces most of the country's grains. Type I Allergic Hypersensitivity to Poaceae Family pollens in Bronchial Asthma patients in Costa Rica was higher than expected.

Adolescent↗

PubMed1994

[Type I hypersensitivity to gramineae pollen (by species) in allergic rhinitis patients].

A total of 184 Costa Rican Allergic Rhinitis patients with at least one positive skin prick test in a previous study were tested for reaction to Poaceae tribes and species compared with 100 non atopic subjects. Skin prick tests were applied for the most abundant species present in Costa Rica. Highest positivity rates among the Allergic Rhinitis patients were obtained with Anthoxatum odoratum (83.2%) (Tribe Aveneae), Panicum maximum (82.1%) (Paniceae), Panicum mole (78.3%) (Paniceae) and Holcus lanatus (77.7%) (Aveneae). The only species with a low percentage of positivity was Uniola pittieri. There was a high prevalence of positive prick tests to pollens of the Poaceae species used for food, including corn, sorghum, sugar cane and rice. All exceeded 60% of positive results in the patients (against 2% in controls). These cultivated species have a special pattern of flowering which can explain the known exhacerbation of allergic symptoms in patients with perennial allergic rhinitis.

Adolescent↗

PubMed1994

[Correlation of allergy positivity for the tests of gramineas between tribes and species].

In a sample of 190 Costa Rican allergic patients and 100 non atopic subjects there was a positive correlation of positivities in skin prick tests to individual Poaceae species and to the Tribe. The exceptions were Panicum maximun, Panicum molle, and Holcus lanatus, because they presented higher individual positivities. A table of correlations among tribes is included. Skin prick tests for Poaceae pollens should be organized by tribes to prevent a 25% of false negative cases.

Adolescent↗

PubMed1990

Isolation and characterization of a new mutant of Saccharomyces cerevisiae with altered synthesis of 5-aminolevulinic acid.

A new gene, RHM1, required for normal production of 5-aminolevulinic acid by Saccharomyces cerevisiae, was identified by a novel screening method. Ethyl methanesulfonate treatment of a fluorescent porphyric strain bearing the pop3-1 mutation produced nonfluorescent or weakly fluorescent mutants with defects in early stages of tetrapyrrole biosynthesis. Class I mutants defective in synthesis of 5-aminolevulinate regained fluorescence when grown on medium supplemented with 5-aminolevulinate, whereas class II mutants altered in later biosynthetic steps did not. Among six recessive class I mutants, at least three complementation groups were found. One mutant contained an allele of HEM1, the structural gene for 5-aminolevulinate synthase, and two mutants contained alleles of the regulatory gene CYC4. The remaining mutants contained genes complementary to both hem1 and cyc4. Mutant strain DA3-RS3/68 contained mutant gene rhm1, which segregated independently of hem1 and cyc4 during meiosis. 5-Aminolevulinate synthase activity of the rhm1 mutant was 35 to 40% of that of the parental pop3-1 strain, whereas intracellular 5-aminolevulinate concentration was only 3 to 4% of the parental value. Transformation of an rhm1 strain with a multicopy plasmid containing the cloned HEM1 gene restored normal levels of 5-aminolevulinate synthase activity, but intracellular 5-aminolevulinate was increased to only 9 to 10% of normal. We concluded that RHM1 could control either targeting of 5-aminolevulinate synthase to the mitochondrial matrix or the activity of the enzyme in vivo.

5-Aminolevulinate Synthetase↗