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Biomedical subjects

F Mendez

Publications and source records attributed to F Mendez.

At least 19 recordsLinked to original sources
PubMed2005

[Stress, hemodynamic and immunological responses to inhaled and intravenous anesthetic techniques for video-assisted laparoscopic cholecystectomy].

OBJECTIVE: Given the immunomodulatory effects of anesthesia and surgery, 2 anesthetic regimens in clinical use were compared to evaluate hemodynamic, stress, and immunologic response in patients undergoing laparoscopic cholecystectomy. PATIENTS AND METHODS: Randomized controlled trial in patients classified ASA I and scheduled for laparoscopic cholecystectomy. Patients were randomly assigned to the inhaled anesthetic group (13 anesthetized with propofol-fentanyl-isoflurane) or the total intravenous anesthesia (TIVA) group (14 patients anesthetized with propofol-remifentanil). Patients in both groups received the muscle relaxant vecuronium. We assessed hemodynamic variables, cortisol levels, prolactin, interleukin 6, white cell and lymphocyte counts before, during (1 hour after induction) and after (24 hours and 7 days) surgery. RESULTS: Hemodynamic variables were stable in both groups. Significant changes in prolactin levels and markers of immune and inflammatory responses between baseline and later measurements occurred in both groups. Patients who received TIVA had no change in cortisol levels at any time during the study. The TIVA group had lower levels of cortisol than did the inhaled anesthesia group (TIVA, 207 [SD, 100] ng/mL; inhaled 293 [97] ng/mL; P<0.05)), higher neutrophil counts (TIVA, 75 [12.5]%; inhaled: 62 [20]%; P<0.05) and higher CD4+ T lymphocyte counts (TIVA, 53 [11.6]%; inhaled: 42 [17.6]%; P<0.001). CONCLUSION: Although both techniques afford hemodynamic stability, lower cortisol levels were observed with the application of TIVA with propofol-remifentanil. That would be the technique of choice for patients with compromised immune response.

Adult↗
PubMed2000

Heat shock protein 70 binds to human apurinic/apyrimidinic endonuclease and stimulates endonuclease activity at abasic sites.

The interaction of human heat shock protein 70 (HSP70) with human apurinic/apyrimidinic endonuclease (HAP1) was demonstrated by coimmunoprecipitation. A combination of HSP70 and HAP1 also caused a shift in the electrophoretic mobility of a DNA fragment containing an apurinic/apyrimidinic site. The functional consequence of the HSP70/HAP1 interaction was a 10-100-fold enhancement of endonuclease activity at abasic sites. The physical and functional interaction between HSP70 and HAP1 did not require the addition of ATP. The association of HSP70 and a key base excision repair enzyme suggests a role for heat shock proteins in promoting base excision repair. These findings provide a possible mechanism by which HSP70 protects cells against oxidative stress.

Base Sequence↗
PubMed2000

Heat-shock proteins associated with base excision repair enzymes in HeLa cells.

Two enzymes of base excision repair (BER), uracil DNA glycosylase (UDG) and DNA polymerase beta (beta pol), from HeLa cells co-eluted from Superose 12 FPLC columns. The UDG was completely displaced from 150-180-kDa fractions to 30- 70-kDa fractions by brief treatment with 0.5 N NaCl, pH 3.0, as expected when protein-protein associations are disrupted, but beta pol was not displaced by this treatment. UDG was not essential to the presence of beta pol in the 150-180-kDa enzyme complex. beta pol and UDG apparently reside in separate but co-eluting structures. Immunoaffinity chromatography showed that the association of UDG and beta pol was accounted for by attachment in common to DNA and that the association was abolished by eliminating DNA. Evidence for base excision repairosomes containing UDG and beta pol in protein-protein assemblies was not found. However, UDG and human AP endonuclease (HAP1) were associated with HSP70 and HSP27, which are present in 150-180-kDa and 30-70-kDa proteins of cell sonicates. The association of HSPs with BER enzymes was confirmed by hydroxyl radical protein-protein footprinting and immunoaffinity tests. The association of HSPs and BER enzymes is a novel finding. HSP binding may account for the presence of BER enzymes in the two large size class fractions and HSPs may have functional roles in BER.

Chromatography, Affinity↗
PubMed1999

Accelerated regression of brain metastases in patients receiving whole brain radiation and the topoisomerase II inhibitor, lucanthone.

PURPOSE: To determine if lucanthone crossed the blood-brain barrier in experimental animals; and to determine accelerated tumor regression of human brain metastases treated jointly with lucanthone and whole brain radiation. METHODS AND MATERIALS: The organ distribution of 3H lucanthone in mice and 125I lucanthone in rats was determined to learn if lucanthone crossed the blood-brain barrier. Size determinations were made of patients' brain metastases from magnetic resonance images or by computed tomography before and after treatment with 30 Gy whole brain radiation alone or with lucanthone. RESULTS: The time course of lucanthone's distribution in brain was identical to that in muscle and heart after intraperitoneal or intravenous administration in experimental animals. Lucanthone, therefore, readily crossed the blood-brain barrier in experimental animals. CONCLUSION: Compared with radiation alone, the tumor regression in patients with brain metastases treated with lucanthone and radiation was accelerated, approaching significance using a permutation test at p = 0.0536.

Animals↗
PubMed1999

Radiolytic and photodynamic modifications of ion transport through the plasma membrane of OK cells: a comparison.

PURPOSE: This study seeks to compare functional modifications of the plasma membrane induced either by water radiolysis (80 kV X-rays) or by visible light in the presence of the photosensitizer photofrin II. The two methods generate different kinds of free radicals and other reactive species such as singlet oxygen. Their effect on the electrical properties of the plasma membrane is investigated. METHOD: The patch-clamp technique (whole-cell and inside-out configuration) was applied to the plasma membrane of an epithelial cell line (opossum kidney (OK) cells). RESULTS: Generation of the reactive species gave rise to a decay of the membrane potential, to an inactivation of K+-channels, and to an increase of the leak conductance of the membrane. All three effects were strongly involved in photomodification of the membrane. Following X-ray exposure (doses up to 1000 Gy) on the other hand, the decay of the membrane conductance caused by inactivation of ion channels was predominant, while the increase of the leak conductance and the depolarization were comparatively minor responses. CONCLUSION: Photomodification of the plasma membrane by visible light in the presence of the membrane-active sensitizer photofrin II is of considerably greater consequence for the electrical properties of the membrane, compared with exposure to ionizing radiation of the membrane and its aqueous environment. The differences are thought to reflect the types of reactive species produced by the two methods and their site of generation.

Animals↗
PubMed1998

Near-visible ultraviolet light induces a novel ubiquitous calcium-permeable cation current in mammalian cell lines.

1. We studied the immediate and short-term effects of UV light in the near-visible range at the cellular and membrane level using the whole-cell patch-clamp technique in combination with digital fluorescence imaging. 2. Illumination with monochromatic UVA light (340-380 nm) induced a sustained non-saturable increase in membrane conductance dependent on wavelength and light intensity in several different mammalian cell types including RBL, mast, HEK, PC12 and 3T3 cells. 3. The current was non-selective for cations and permeable to Ca2+, but was inhibited by trivalent cations and was not due to the activation of an endogenous ion channel. We termed this novel current ILiNC for light-induced non-selective cation current. 4. A similar current was evoked by chemical peroxidants such as hydrogen peroxide and tertbutylhydroperoxide, but not by cytosolic oxidized glutathione. 5. The free-radical scavengers tocopherol (vitamin E) and ascorbic acid (vitamin C) significantly reduced the UV light effect. 6. The generation of the current was membrane delimited since it could be induced by the same UVA treatment in cell-free membrane patches showing a similar wavelength dependence. 7. These results suggest that ILiNC is activated by UVA light-induced generation of free radicals acting through lipid or protein peroxidation, and may represent a ubiquitous mechanism by which Na+ and Ca2+ can enter cells after phototoxic or free radical-induced membrane damage.

Animals↗
PubMed1997

Topoisomerase inhibition by lucanthone, an adjuvant in radiation therapy.

PURPOSE: To determine whether lucanthone can inhibit human topoisomerases in vitro. METHODS AND MATERIALS: Lucanthone was incubated with human topoisomerases II and I together with their plasmid substrates, to determine if lucanthone interfered with the catalytic activities of topoisomerases and if it enhanced the formation of DNA strand breaks, as determined by agarose gel electrophoresis of the resultant plasmid forms. RESULTS: Incubation of the enzymes with lucanthone inhibited the catalytic activity of topoisomerases II and I. With topoisomerase II, it increased the abundance of DNA double strand breaks (cleavable complexes). CONCLUSION: Lucanthone, like actinomycin D, inhibited topoisomerases II and I. It may act to enhance the yield of DNA double strand breaks in cells through a mechanism of topoisomerase II inhibition.

Antineoplastic Agents, Phytogenic↗
PubMed1996

Protein-protein interactions between the Escherichia coli single-stranded DNA-binding protein and exonuclease I.

It was demonstrated previously that a deoxyribophosphodiesterase (dRpase) activity is associated with the DNA repair enzyme exonuclease I, and that this activity is stimulated by the addition of the E. coli single-stranded DNA-binding protein (Ssb). This activity catalyzes the release of deoxyribose-phosphate groups at apurinic/apyrimidinic (AP) sites in the DNA that have been cleared by the action of an AP endonuclease. We have now used the yeast two-hybrid system to demonstrate that a protein-protein interaction occurs between exonuclease I and Ssb. When the E. coli ssb gene was fused in frame to the DNA-activating domain of the GAL4 transcriptional activator and the exonuclease I gene was fused in frame to the DNA-binding domain, a functional GAL4 transcriptional activator was produced as determined by growth of yeast on selective medium and the measurement of beta-galactosidase activity. We have also demonstrated that Ssb can stimulate the dRpase activity of exonuclease I using double-stranded bacteriophage M13 DNA containing several strand interruptions at incised AP sites. These results suggest that Ssb may be required for efficient base-excision repair in bacteria.

Bacteriophage M13↗
PubMed1995

Cryptosporidium parvum oocyst antigens recognized by sera from infected asymptomatic adult cattle.

Gel electrophoresis and Western blotting were used to investigate the recognition of Cryptosporidium parvum oocyst antigens by sera from asymptomatic C. parvum-seropositive cattle with or without coccidian oocysts in their faeces (C. parvum or/and Eimeria spp.). Most sera from coprologically C. parvum-positive animals (71%) recognized fractions in the 17-20 kDa range; these fractions were not recognized by sera from coprologically negative animals. In addition, most sera with antibodies to C. parvum (whether from coprologically positive or negative animals) showed moderate or strong reaction with antigenic fractions in the 47-49.5 kDa range (43%) and the 56.5-69 kDa range (80%).

Animals↗
PubMed1994

Enhanced repair endonuclease activities from radiation-arrested G2 phase mammalian cells.

HeLa cells arrested in G2 phase 22 h after receiving 11.5 Gy gamma-radiation contained 3.6-fold more EDTA-resistant DNA repair endonuclease activity than unirradiated cells. Enzyme activity was determined by measuring the release of fragments from an irradiated repetitive alpha DNA substrate or from synthetic substrates containing a single modified base, 8-oxoguanine (8-oxo-G), a major radiation product. It appeared that the radiation-induced enhanced repair activity in some cells might be a feature of radiation-induced G2 arrest. Indeed, unirradiated G2 HeLa cells that had been synchronized by double thymidine block contained 3-7-fold more endonuclease activity than G1 or S-phase cells. Similarly, two of four other cell lines tested exhibited elevated repair endonuclease activity in G2. However, all six cell lines tested exhibited radiation-enhanced repair endonuclease activity. Therefore, the underlying mechanism for radiation enhancement of enzyme activity remains to be clarified and does not seem to be completely accounted for as a consequence of G2 arrest. The results showed different substrate specificities among cell lines as well as differences during the cell cycle of individual cell lines. Repair endonuclease activity from all cell lines which we have tested were associated with 60-70 kDa proteins from Superose 12 columns. Since reports from other laboratories have described several different DNA repair activities in 50-70 kDa Superose 12 fractions, it seems possible that the DNA repair enzymes may be associated in a repairosome structure.

Animals↗
PubMed1992

Repair of ionizing radiation damage in primate alpha DNA transfected into rat cells.

The time-course of repair of irradiated primate alpha DNA was studied after transfection and recovery from rat NRK cells. Rat cells were chosen for transfection because they have no alpha DNA. Plasmid pBUC4 alpha 10, containing 10 tandem 172 bp alpha DNA subunits in its 5 kbp DNA, was irradiated and introduced into the rat cells by electroporation. The transfected alpha DNA was then recovered from NRK nuclei free of extraneous rat DNA, permitting study of the fate of the transfected alpha DNA in time-course experiments. alpha DNA continuously entered nuclei for processing in the first 2.5 h after transfection. The pool of damaged bases in alpha DNA in NRK nuclei was detectable 2.5 h after transfection. Radiation-induced alpha DNA fragments of electrophoretic mobility intermediate between those of unit nucleotide length were prominent in sequencing gel analyses of alpha DNA for 5-150 min after transfection. These intermediate mobility fragments initially disappeared with T 1/2 of 6-20 min. The alpha DNAs of intermediate mobility are presumed to be intermediates in DNA repair. Residual DNA base damage which had not been processed in the transfected cells could later be unmasked in vitro by conversion to strand breaks by beta-elimination using heat and piperidine or endonuclease III of E. coli. Irradiation of the recipient NRK cells with 5 Gy 4 hours before transfection prolonged the time during which intermediate mobility species could be found, consistent with the increased frequency of intermediate mobility species observed in DNA of monkey CV-1 cells pretreated with small doses of radiation before 300 Gy (Bases et al. 1990).

Animals↗
PubMed1992

Enhanced excision repair activity in mammalian cells after ionizing radiation.

Monkey CV-1 cells which had received 5 Gy 12 h before harvesting lysates from their cell cultures contained approximately three times as much DNA excision repair enzyme activity as unirradiated cells. The activity was determined in crude cell lysates by the release of intermediate mobility DNA fragments and fragments with 3'-phosphoryl ends from 5'-32P-end labelled irradiated 95 bp alpha DNA. Different 3'-termini endow the fragments with differing mobilities, signifying steps in the processing of radiation damaged DNA. Similar results were obtained when Krebs II mouse tumour cells growing in mice as ascites received 5 Gy 12 h before harvest. The enzyme activities from CV-1 cells and from Krebs II cells were partially purified as 60-70 kDa proteins on Superose 12 or Ultrogel AcA-54 columns. Divalent cations were not required for enzyme activity. A 23 nucleotide long defined duplex oligodeoxynucleotide substrate containing a single 8-oxodG residue was also very actively cleaved by the partially purified cell enzymes. 8-oxoguanine is a major product of ionizing radiation's action on DNA and was recognized by the enzymes described here. The mechanism by which radiation increased excision repair activity of cellular enzymes is not understood.

Animals↗
PubMed1990

Serotype-specific outbreak of group B meningococcal disease in Iquique, Chile.

From 1979 to August 1987, there have been 178 cases of meningococcal disease in Iquique, Chile, a city of about 140,000. The attack rate for the last 5 years has been in excess of 20/100,000 per year, more than 20 times greater than for the country overall. The mortality rate was 6%. The disease occurred in patients with ages from 4 months to 60 years, but 89% of cases were in patients less than 21 years. The largest number of cases were in the age group 5-9 years (n = 54), but the highest incidence occurred in children less than 1 year of age (72.8/100,000 per year). The male/female ratio was 1.2. Cases occurred all year round with little seasonal variation. Of the 178 cases, 173 were biologically confirmed. Serogroup analysis of strains from 135 patients revealed A = 1, B = 124, C = 10. Forty-four group B strains from 1985-7 were serotyped: 15:P1.3 = 36, 15:NT = 4, 4:P1.3 = 2, NT:NT = 2. Ten of 11 of the outbreak strains tested were sulfadiazine-resistant. This is the first recognized outbreak caused by a Gp B:15 strain in South America. It shares many of the characteristics of outbreaks caused by closely related strains in Europe, such as a predilection for older children and adolescents, sulfadiazine-resistance, and sustained high attack rates. The Iquique strain (B:15:P1.3) belongs to the same genetic clone (ET-5 complex) as the Norway (B:15:P1.16) and the Cuban (B:4:P1.15) strains.

Adolescent↗
PubMed1990

DNA base and strand damage in X-irradiated monkey CV-1 cells: influence of pretreatment using small doses of radiation.

Base damage in alpha DNA from irradiated monkey CV-1 cells was determined by measuring release of 5'-32P-end labelled DNA fragments after digestion with endonuclease III of E. coli. The frequency and base sequence locations of the enzyme-sensitive sites were determined. Fragments were released from irradiated DNA at sequence sites of pyrimidines and guanines. The time for repair of half the single strand breaks was approximately 1.5 h. Repair of base damage as judged from loss of enzyme-sensitive sites in DNA was slower, with more than half of the damaged bases still detectable after 4 h of repair. Two important changes in the pattern of fragment release from DNA were produced when small radiation doses preceded the large ones needed to produce measurable DNA strand breaks and base damage. 5 Gy to cells incubated several hours before 320 Gy increased by five-fold the abundance of small DNA fragments with 3'-phosphoryl termini detected in high-resolution denaturing gels. These increases were detectable with doses as small as 0.2 Gy and were accompanied by the appearance of new species of DNA fragments of intermediate mobility at specific locations in the base sequence. The patterns resemble those produced by digesting DNA from heavily irradiated cells with endonuclease III.

Animals↗
PubMed1988

Base sequence damage in DNA from X-irradiated monkey CV-1 cells.

Two kinds of 3'-ends were detected in DNA scission fragments of highly repetitive primate component alpha DNA which were isolated from irradiated monkey CV-1 cells. The fragments' 3'-ends were characterized by 5'-32P-end labelling the DNA, followed by examination in high-resolution polyacrylamide gels under denaturing conditions. Hydrolysis of the labelled fragments' termini with exonuclease III of E. coli or by the 3'-phosphatase activity of T4 polynucleotide kinase generated a third, slowest migrating species in each mobility size class. Reference to mobility size class standards makes it highly probable that the fragment ends generated by X-rays in cells are 3'-phosphoryl and 3'-phosphoglycolate, and that they are converted to slower migrating fragments with 3'-OH ends, similar to results obtained with DNA irradiated in water (Henner et al. 1982, 1983 a, b). Densitometer measurements of gel autoradiograms showed that X-ray induction of DNA fragments with 3'-phosphoryl and 3'-phosphoglycolate ends was dose-dependent over a range 100-900 Gy. In CV-1 cells the frequency of single-strand breaks in alpha DNA was 8.6 x 10(-7) breaks/nt/Gy. The two kinds of ends disappeared in post-radiation incubation with a half-time of 1.6 h. These results provide a new means to study X-ray damage and repair of specific sequences in animal cells.

Animals↗
PubMed1987

Expression of prokaryotic genes after transfection of X-irradiated plasmids into primate cells.

Expression of the prokaryotic gene for chloramphenicol acetyltransferase (EC 2.3.1.28) (CAT) in primate cells transfected with X-irradiated plasmid pSV2CAT was determined in transient expression assays. CAT expression did not depend upon the presence of supercoiled plasmids, but relaxed circular forms were essential. X-ray conversion of relaxed circles to linear forms paralleled the loss of CAT expression, with identical D0's in the first part of dose-response curves. X-ray-induced loss of supercoiled forms was complete at much lower doses. The D0 for inactivation of CAT expression by X irradiation of the plasmids in 1 mM Tris buffer was 270 Gy; it was 13 Gy for plasmids irradiated in water. The D0's for conversion of pSV2CAT to relaxed circle forms were only one-seventh as large as the D0's for CAT inactivation after X-ray in water or in 1 mM Tris buffer. Expression of the CAT gene in some representative repair-deficient human fibroblasts transfected with X-irradiated pSV2CAT was less than in monkey CV-1 cells or cell lines from normal human subjects. These results demonstrate a novel means to study low levels of X-ray damage in DNA correlating specific X-ray damage in the DNA with expression of the gene in unirradiated primate cells.

Acetyltransferases↗