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Biomedical subjects

H Flores

Publications and source records attributed to H Flores.

At least 19 recordsLinked to original sources
PubMed2005

Mediterranean and Amerindian MHC class II alleles are associated with multiple sclerosis in Mexicans.

OBJECTIVES: Human leukocyte antigen (HLA)-DRB1, DQA1, DQB1 allele typing was performed in Mexicans Mestizos with multiple sclerosis (MS) to define the HLA class II alleles associated with the disease in this population. METHODS: Patients (n = 51) diagnosed according to the Poser criteria and a group of 173 unrelated healthy subjects were studied. PCR-SSOP and PCR-SSP were used for genotyping. RESULTS: Fifty five percent of the patients were females. The mean age at disease onset was 27 years. A relapsing-remitting disease was the most frequent type of MS (67%). A significant association of DRB1*0403 (OR = 5.68) with MS was shown. DRB1*0802 was also involved in susceptibility (OR = 2.41). An excess of DRB1*0802 homozygotes was observed in patients (P = 0.005), this genotype being in genetic equilibrium in controls. CONCLUSIONS: Two novel class II associations are described in Mexicans with MS: DRB1*0403 and DRB1*0802. Both alleles share with DRB1*1501, valine-86 and negatively charged amino acids, in the DRB1-anchoring motif of pocket 4.

Adolescent↗
PubMed2004

Detection of vitamin A deficiency in Brazilian preschool children using the serum 30-day dose-response test.

BACKGROUND: Vitamin A deficiency (VAD) is endemic in Brazil and health professionals have difficulty in recognizing its subclinical form. In addition, serum retinol concentrations do not always represent vitamin A status in the organism. OBJECTIVE: To identify VAD in preschool children by the serum 30-day dose-response test (+S30DR) and to examine its potential as a tool for the assessment of vitamin A status in the community. DESIGN: A prospective transverse study in which blood samples were obtained from 188 preschool children for the determination of serum retinol concentrations and the children were submitted to ocular inspection and anthropometric measurements. Information about the presence of diarrhea and/or fever during the 15 days preceding the study was also obtained. The children received an oral dose of 200,000 IU vitamin A immediately after the first blood collection. A second blood sample was obtained 30-45 days after supplementation in order to determine the +S30DR. RESULTS: In all, 74.5% (140/188; 95% confidence interval: 68.3-80.7%) of the children presented +S30DR values indicative of low hepatic reserves. The mean serum retinol concentration was significantly lower before supplementation (0.92 and 1.65 micromol/l, respectively; P < 0.0001). No child presented xerophthalmia; 3.7% (7/188) of the children were malnourished. The presence of fever and/or diarrhea during the 15 days preceding the first blood collection did not affect the +S30DR value. CONCLUSIONS: The prevalence of VAD in the study group was elevated. +S30DR proved to be a good indicator of subclinical VAD in children from an underdeveloped country.

Anthropometry↗
PubMed2000

Reciprocal domain evolution within a transactivator in a restricted sequence space.

offhough the concept of domain merging and shuffling as a major force in protein evolution is well established, it has been difficult to demonstrate how domains coadapt. Here we show evidence of coevolution of the Sinorhizobium meliloti NifA (SmNifA) domains. We found that, because of the lack of a conserved glycine in its DNA-binding domain, this transactivator protein interacts weakly with the enhancers. This defect, however, was compensated by evolving a highly efficient activation domain that, contrasting to Bradyrhizobium japonicum NifA (BjNifA), can activate in trans. To explore paths that lead to this enhanced activity, we mutagenized BjNifA. After three cycles of mutagenesis and selection, a highly active derivative was obtained. Strikingly, all mutations changed to amino acids already present in SmNifA. Our artificial process thus recreated the natural evolution followed by this protein and suggests that NifA is trapped in a restricted sequence space with very limited solutions for higher activity by point mutation.

Amino Acid Sequence↗
PubMed1999

[Nutritional status of vitamin A in mother/newborn pairs from 2 hospital nurseries in Rio de Janeiro, Brazil].

The aim of the present study was to assess maternal and newborn (umbilical blood) vitamin status in 220 mothers/newborns at birth from two public health centers in Rio de Janeiro, Brazil. The proportion of low retinol levels (cut-off point 1.05 mumol/L) in the umbilical cord of newborns (55.4%) was greater than found in their mothers (23.6%). A highly significant correlation (X2 = 14.2; p < 0.0001) was found between the levels of retinol of mothers and newborns. The overall prevalence of low levels of retinol in the mother was 23.6% whereas that of newborns was 55.4%. Umbilical cord mean concentration less than 1.05 mumol/L was significantly lower (2.49 +/- 1.08 mumol/L) in mother with vitamin A deficiency (3.21 +/- 0.97 mumol/L; p < 0.0001). Low birth weight was associated with vitamin A deficiency (X2 = 6.86; p < 0.01). These data reinforce the need for close pre-natal attention in vitamin A status.

Adult↗
PubMed1997

Temperature-sensitive mutants of the EcoRI endonuclease.

The EcoRI endonuclease is an important recombinant DNA tool and a paradigm of sequence-specific DNA-protein interactions. We have isolated temperature-sensitive (TS) EcoRI endonuclease mutants (R56Q, G78D, P90S, V97I, R105K, M157I, C218Y, A235E, M255I, T261I and L263F) and characterized activity in vivo and in vitro. Although the majority were TS for function in vivo, all of the mutant enzymes were stably expressed and largely soluble at both 30 degrees C and 42 degrees C in vivo and none of the mutants was found to be TS in vitro. These findings suggest that these mutations may affect folding of the enzyme at elevated temperature in vivo. Both non-conservative and conservative substitutions occurred but were not correlated with severity of the mutation. Of the 12 residues identified, 11 are conserved between EcoRI and the isoschizomer RsrI (which shares 50% identity), a further indication that these residues are critical for EcoRI structure and function. Inspection of the 2.8 A resolution X-ray crystal structure of the wild-type EcoRI endonuclease-DNA complex revealed that: (1) the TS mutations cluster in one half of the globular enzyme; (2) several of the substituted residues interact with each other; (3) most mutations would be predicted to disrupt local structures; (4) two mutations may affect the dimer interface (G78D and A235E); (5) one mutation (P90S) occurred in a residue that is part of, or immediately adjacent to, the EcoRI active site and which is conserved in the distantly related EcoRV endonuclease. Finally, one class of mutants restricted phage in vivo and was active in vitro, whereas a second class did not restrict and was inactive in vitro. The two classes of mutants may differ in kinetic properties or cleavage mechanism. In summary, these mutations provide insights into EcoRI structure and function, and complement previous genetic, biochemical, and structural analyses.

Bacteriophage lambda↗
PubMed1997

Isolated domain II and III from the Bacillus thuringiensis Cry1Ab delta-endotoxin binds to lepidopteran midgut membranes.

The DNA fragment encoding Cry1Ab domain II-III (45.3 kDa) was cloned and expressed. Domain II-III is expressed in low yields. In vitro binding analysis to Manduca sexta and Trichoplusia ni larval midgut tissue sections demonstrated that domain II-III fragment bound along the microvilli of the midgut epithelium, indicating that this fragment retains binding functionality in the absence of domain I. Binding of domain II-III to the midgut brush border membrane proteins from T. ni larvae indicated that Cry1Ab toxin and domain II-III bind to the same 150 kDa protein. In contrast, in M. sexta membranes, Cry1Ab toxin binds to 200 and 120 kDa proteins, and domain II-III only binds to the 200 kDa protein. Finally, binding assays with isolated brush border membrane vesicles showed that the interaction of domain II-III with the membrane vesicles is highly reversible, supporting the proposition that the integration of domain I into the membrane could participate in the irreversible binding of the toxin. These studies confirm that this part of the toxin is involved in binding interactions and could be separated as a discrete fragment that conserves at least part of its functionality.

Animals↗
PubMed1995

Saturation mutagenesis of His114 of EcoRI reveals relaxed-specificity mutants.

EcoRI recognizes and cleaves DNA at GAATTC sites and is one of the best characterized sequence-specific restriction endonucleases (ENases). In previous studies, an EcoRI mutant, which exhibited relaxed substrate specificity and cleaved both canonical and EcoRI star sites, was isolated. This mutant enzyme has Tyr instead of His114. Here, we subjected residue 114 of the EcoRI ENase to saturation mutagenesis. The resulting mutant enzymes were characterized both in vivo and in vitro, resulting in the identification of mutants with canonical (H114K, Q, D, I) or relaxed (H114Y, F, S, T) specificity, as well as one mutant with severely impaired activity (H114P). In the X-ray structure of an EcoRI-substrate complex, His114 is located between the catalytic and recognition regions of EcoRI and may directly contact the DNA phosphate backbone. Based on our genetic and biochemical findings and the X-ray structure, we propose that His114 participates in substrate recognition and catalysis, either directly, via protein-DNA interactions, or indirectly, by mediating conformational changes that trigger DNA cleavage in response to substrate recognition.

Amino Acid Sequence↗
PubMed1994

Microbial systems and directed evolution of protein activities.

Recent advances in recombinant DNA methodology have had an important impact on the capacity to manipulate protein-coding sequences. The appearance of new, powerful screening systems completes a scenario for conducting directed evolution experiments. We review here some of the latest developments in experimental approaches to directed evolution, utilizing microbial systems. These include phage display, surface display, operator-repressor systems, and novel mutagenesis approaches. We also highlight the achievements and limitations of current methodologies. We present strategies used by our own group that permitted isolation of specificity mutants of beta-lactamase. Possible improvements for the future of the variation-selection approach to the study and manipulation of proteins are presented.

Bacteria↗
PubMed1994

[The psychological factors that affect the making of a decision for surgical sterilization and the likely later changes].

The practice of female surgical sterilization in Panama during the past decades, motivated the realization of this study, to determine the factors which caused women to use this permanent contraceptive method, and to see if regrets later occurred after that decision, in the sterilized women. A sample of 151 women were selected, between the ages of 28 and 45 years, concurrently pregnant and with 2 or more living children at the moment of the study. Psychological tests were applied during three different phases (pre-operatively and subsequently 12 and 24 months, post operatively). Results showed that female sterilization did not cause long term complications, and amongst the majority of women conforming the sample, a stable psychological profile was found up to 24 months after the interventions, with a significant improvement in the last evaluation of the social adaptation variable, in comparison to the pre-operative assessment.

Adult↗
PubMed1993

Rat models of protein and protein-energy malnutrition.

Two animal models of infantile malnutrition were produced where some aspects of carbohydrate metabolism can be studied: protein-depleted (PD, "kwashiorkor") rats, fed a protein-free diet for 18 days, starting 3 days before weaning, and chronically malnourished animals during suckling (PED, "marasmus") where 20-22 pups were fed by the same dam. PED and PD rats were sacrificed at 21 and 36 days of age, respectively. Body weight was 35% in PD, and 50% in PED groups when compared to their age-matched controls. After an overnight fast, some of the PD animals presented with low blood glucose levels, and the levels of lactic and pyruvic acids were depressed in PED, while lactic acid was increased in PD animals. Blood glucose utilization was reduced in PED, and increased in PD rats, while the opposite was true for the kidney gluconeogenic capacity. When compared to the respective control values, muscle glycogen was high in both groups, while the level of liver glycogen was unchanged. The rate of muscle glycolysis was decreased in both groups. These results are in agreement with clinical observations, suggesting that these models may be used to study some of the metabolic changes brought about by infantile malnutrition.

Animals↗
PubMed1991

Combinatorial mutagenesis of three major groove-contacting residues of EcoRI: single and double amino acid replacements retaining methyltransferase-sensitive activities.

A library of mutant ecoRIR genes encoding EcoRI restriction endonuclease was generated using trinucleotide blocks and a combination of recombinant DNA procedures, including primer extension and the polymerase chain reaction. Codons corresponding to three amino acids (E144, R145 and R200), previously implicated in the specific recognition of the DNA substrate, were combinatorially mutated so as to generate a library that potentially contains all 20(3) possible single, double and triple aa replacements, in a balanced distribution. Inspection of the phenotypes of Escherichia coli colonies bearing the mutant genes showed that several of them retained activities that were deleterious to the cells but were still protected by the EcoRI methyltransferase. These included new enzyme variants, including non-conservative single (Thr or Val for Glu144) and double (Val for Glu144 and Thr for Arg145) replacements.

Amino Acids↗
PubMed1991

Serum vitamin A distribution curve for children aged 2-6 y known to have adequate vitamin A status: a reference population.

Serum vitamin A was determined before and 30-45 d after the administration of 60.6 mg (212 mumol) vitamin A to 544 Brazilian children residing in slum areas of Recife. The frequency-distribution curves were compared in a subgroup of children whose vitamin A status was assessed initially by the relative-dose-response (RDR) test. The curves of children with negative (adequate status) and positive (inadequate status) RDR tests were different. The difference disappeared after supplementation. The shape of the distribution curve after supplementation was close to normal with a mean, median, and 95% confidence interval of 1.78 +/- 0.49, 1.68, and 1.02-2.90 mumol/L, respectively. The postsupplementation curve derived from this underprivileged child population may serve as a reference for diagnostic, surveillance, and program-evaluation purposes.

Brazil↗
PubMed1990

[Trans-laparoscopic intratubal insemination].

Intratubal insemination was practiced in 21 patients during laparoscopy either for infertility study or second look laparoscopy for medically treated endometriosis, stimulation of ovulation was performed during the menstrual cycle with clomiphene for five days, at the sixth day of the cycle we start the administration of human menopausal gonadotrophins. Ovulation was monitored with ultrasound and estradiol determinations. From 30 to 36 hours before laparoscopy chorionic gonadotrophin was administrated, homologous capacitated sperm were introduced directly in the fallopian tubes results in two pregnancies (9%). One terminated in abortion for the first trimester, the other resulted in a normal pregnancy with birth at term.

Adult↗
PubMed1990

[Translaparoscopic intratubal insemination].

Intratubal insemination was practiced in 21 patients during laparoscopy either for infertility study or second look laparoscopy for medically treated endometriosis, stimulation of ovulation was performed during the menstrual cycle with clomiphene for five days, at the sixth day of the cycle we start the administration of human menopausal gonadotrophins. Ovulation was monitored with ultrasound and estradio determinations. From 30 to 36 hours before laparoscopy corionic gonadotrophin was administrated, homologous capacitated sperm was introduced directly in the fallopian tubes results in two pregnancies (9%). One terminated in abortion for the first trimester, the other resulted in a normal pregnancy with birth at term.

Adult↗