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M Ayala

Publications and source records attributed to M Ayala.

At least 19 recordsLinked to original sources
PubMed2002

Toxicity of ultraviolet radiation exposure to the lens expressed by maximum tolerable dose.

The maximum tolerable dose (MTD2.3:16) for avoidance of cataract on exposure to ultraviolet radiation (UVR)-300 nm in the rat was here estimated at 3.65 kJ/m2. Sprague-Dawley rats were unilaterally exposed to UVR in the 300 nm wavelength region. One week after the exposure, the intensity of forward light scattering was measured. Toxicity for continuous response events can be estimated with MTD. Current safety standards for avoidance of cataract after exposure to UVR are based on a binary response event. It has, however, recently been shown that UVR-induced cataract is a continuous dose-dependent event. MTD provides a statistically well-defined criterion of toxicity for continuous response events.

Animals↗
PubMed2001

Biocatalytic chlorination of aromatic hydrocarbons by chloroperoxidase of Caldariomyces fumago.

Chloroperoxidase from Caldariomyces fumago was able to chlorinate 17 of 20 aromatic hydrocarbons assayed in the presence of hydrogen peroxide and chloride ions. Reaction rates varied from 0.6 min(-1) for naphthalene to 758 min(-1) for 9-methylanthracene. Mono-, di- and tri-chlorinated compounds were obtained from the chloroperoxidase-mediated reaction on aromatic compounds. Dichloroacenaphthene, trichloroacenaphthene, 9,10-dichloroanthracene, chloropyrene, dichloropyrene, dichlorobiphenylene and trichlorobiphenylene were identified by mass spectral analyses as products from acenaphthene, anthracene, pyrene and biophenylene respectively. Polycyclic aromatic hydrocarbons with 5 and 6 aromatic rings were also substrates for the chloroperoxidase reaction. The importance of the microbial chlorination of aromatic pollutants and its potential environmental impact are discussed.

Catalysis↗
PubMed2000

Very high expression of an anti-carcinoembryonic antigen single chain Fv antibody fragment in the yeast Pichia pastoris.

In this paper we report the development of a recombinant strain of the yeast Pichia pastoris, which secretes an anti-carcinoembryonic antigen single chain Fv (scFv) antibody fragment to the culture supernatant as a biologically active protein, at levels of 1.2 g l(-1). The yeast scFv was purified by IMAC, with a final yield of approximately 0.440 g of 93% pure scFv per liter of culture supernatant. The specific activity in ELISA of the yeast scFv was almost three times higher than that of a bacterial periplasmic counterpart. These results reaffirm that the yeast P. pastoris is a suitable host for high level production of scFv antibody fragments with potential in vivo diagnostic and therapeutic applications.

Adenosine Triphosphatases↗
PubMed2000

An action spectrum for UV-B radiation and the rat lens.

PURPOSE: To determine an action spectrum for UV-B radiation and the rat lens and to show the effect of the atmosphere and the cornea on the action spectrum. METHODS: One eye of young female rats was exposed to 5-nm bandwidths of UV-B radiation (290, 295, 300, 305, 310, and 315 nm). Light scattering of exposed and nonexposed lenses was measured 1 week after irradiation. A quadratic polynomial was fit to the dose-response curve for each wave band. The dose at each wave band that produced a level of light scattering greater than 95% of the nonexposed lenses was defined as the maximum acceptable dose (MAD). Transmittance of the rat cornea was measured with a fiberoptic spectrophotometer. The times to be exposed to the MAD in Stockholm (59.3 degrees N) and La Palma (28 degrees N) were compared. RESULTS: Significant light scattering was detected after UV-B at 295, 300, 305, 310, and 315 nm. The lens was most sensitive to UV-B at 300 nm. Correcting for corneal transmittance showed that the rat lens is at least as sensitive to UV radiation at 295 nm as at 300 nm. The times to be exposed to the MAD at each wave band were greater in Stockholm than in La Palma, and in both locations the theoretical time to be exposed to the MAD was least at 305 nm. CONCLUSIONS: After correcting for corneal transmittance, the biological sensitivity of the rat lens to UV-B is at least as great at 295 nm as at 300 nm. After correcting for transmittance by the atmosphere, UV-B at 305 nm is the most likely wave band to injure the rat lens in both Stockholm and La Palma.

Animals↗
PubMed1999

A bacterial single-chain Fv antibody fragment that inhibits binding of its parental anti-E-selectin monoclonal antibody to activated human endothelial cells.

Using the polymerase chain reaction, we cloned, modified, and linked antibody variable (V) region coding genes from a mouse hybridoma, and produced a bacterial single-chain Fv (scFv) antibody fragment specific for E-Selectin. A vector of pBR322 origin, bearing the tryptophan promoter and the ompA bacterial signal peptide, was used to direct scFv expression to periplasm. The vector included a six-histidine coding sequence 5' to the scFv for the purification of the expressed protein using immobilized metal affinity chromatography (IMAC). We found that the VH-Linker-VL 32-33 kDa scFv remained insoluble after cellular fractionation, and transmission electron microscopy showed the new protein to be present in the periplasm as inclusion bodies. The scFv was solubilized using urea, purified using IMAC, and renatured to its active form. In a competitive enzyme-linked immunosorbent assay with activated human vein endothelial cells in the solid phase, the scFv competed for binding with the original monoclonal antibody.

Amino Acid Sequence↗
PubMed1999

High cytoplasmic expression in E. coli, purification, and in vitro refolding of a single chain Fv antibody fragment against the hepatitis B surface antigen.

A single-chain Fv (scFv) antibody fragment against the hepatitis B surface antigen (HBsAg) was expressed in Escherichia coli in the form of two independent fusion proteins, with either 60 ('long') or 27 ('short') amino acid N-terminal encoding sequences related to human interleukin-2. Both fusion proteins were expressed insolubly and at high levels in the bacterial cytoplasm (approximately 30% of total bacterial protein in MM294 cells at a laboratory scale). When recombinant cells were cultured in 5-1 fermentors, expression and optical density increased 2- and 4-fold, respectively, compared to a previous periplasmic insoluble version of the same anti HBsAg scFv. After extraction and solubilization in urea, the cytoplasmic scFvs were purified using immobilized metal ion affinity chromatography, followed by DTT treatment, and refolding by dialysis against a basic pH buffer containing EDTA. The refolded scFvs recognized the recombinant HBsAg in ELISA. Results of an ELISA where antigen affinity chromatography repurified scFvs were used as standards, indicated that refolding efficiencies were high: 56.2% for the 'short' fusion scFv, and 50.6% for the 'long' fusion scFv. Corrected final yields of active scFv were 30.3 and 27.3 mg l-1, respectively, for the aforementioned fusion proteins, 5-6 times better than those reported for the periplasmic scFv variant.

Amino Acid Sequence↗
PubMed1998

The human breast cancer cell line IIB-BR-G has amplified c-myc and c-fos oncogenes in vitro and is spontaneously metastatic in vivo.

IIB-BR-G is an undifferentiated, highly heterogeneous, hormone receptor negative human breast cancer cell line previously established in our laboratory from a patient's primary tumor. An in vitro growing cell line (IIB-BR-G) and a xenotransplanted tumor growing in nude mice (IIB-BR-G(NUDE)) were derived. To further characterize these systems, immunocytochemical analysis was performed for differentiation antigens (PEM 200 kDa, CEA, NCA 90 kDa), blood-group related antigens (Le(x), sTn), oncogenes and tumor suppressor gene products (Her-2/neu protein, p53), metastasis-related cathepsin D and CD63/5.01 Ag, and the chemokine monocyte chemotactic protein 1 (MCP-1). Expression of markers was heterogeneous in these different systems. Previously reported karyotypic analysis has shown extensive chromosomal alterations including double min. Searching for oncogene amplification, we detected augmented copy number of c-myc and c-fos, the last one with two rearranged fragments. No amplification was found for c-erbB-2 in the cell line or in IIB-BR-G(NUDE), although this oncogene was amplified in the patient's primary tumor DNA. The differences observed between the patient's tumor, the cell line and the IIB-BR-G(NUDE) tumors are probably due to clonal expansion of cell variants not present in the original tumor. Electron microscopy of IIB-BR-G growing cells revealed epithelial characteristics with abundant dense granules, presumably secretory, distributed all over the cytoplasm and great nuclear pleomorphism. In vitro, IIB-BR-G cells showed a significant number of invading cells by Matrigel assay. After nearly 40 sequential subcutaneous passages of the original xenograft through nude mice, 80% of recipients developed spontaneous metastases, primarily to the lung and lymph nodes. Since this experimental model allowed to analyze changes produced in cancer cells from the primary tumor during adaptation to in vitro and in vivo growth, our results provide novel insights on the behaviour of hormone independent metastatic breast cancer.

Animals↗
PubMed1997

Acute copper intoxication after a Cu-Ca EDTA injection in rats.

It has been claimed that Cu-Ca EDTA releases the Cu slowly into the blood when injected as a therapeutic agent for Cu deficiency, but when overdosing occurs, animals may die in a few hours after the injection, suggesting that Cu release is faster than expected. A total of 30 rats were divided into six equal groups and injected with Cu-Ca EDTA at 4, 6, 13, 33 or 64 mg/kg body weight, the sixth group acted as control. All rats were sacrificed 24 h later, with the exception of rats from groups 33 and 64 that died 7 and 2 h postinjection respectively. The rate of Cu transference to the liver increased linearly with the dose, from (X +/- S.E.) 14.88 +/- 3.14 in group 4, to 134.47 +/- 21.86 microg/h in group 64. Injected animals developed liver and kidney degeneration, hemorrhage and necrosis, which increased in severity with the Cu dose. Liver Cu accumulation was highest for group 13 (866.8 +/- 84.23 ppm DM), while in group 64 reached only 167.0 +/- 14.99 ppm. It is concluded that during acute Cu poisoning, the rate of transfer is responsible for the liver and kidney damage, rather than the total amount of transferred Cu to those organs, as it is the case in chronic Cu poisoning. The high rate of Cu transfer possibly surpasses the intrinsic mechanisms of Cu deposition and free Cu is left into the cell with the development of necrosis.

Acute Disease↗
PubMed1997

Antigen recognition characteristics and comparative performance in immunoaffinity purification of two monoclonal antibodies specific for the hepatitis B virus surface antigen.

In this paper we describe the antigen recognition characteristics, variable region base and amino acid sequence, and performance as immunoaffinity chromatography ligands of two MAb specific to the alpha determinant of the HBsAg, derived from the same fusion. We show that the epitope recognized by CB-Hep.0 (IgM) is probably associated to an intrachain disulfide bond in the antigen. On the other hand, CB-Hep.1 (IgG2b) recognizes a heat-resistant non-conformation dependent antigenic determinant on HBsAg. PCR-cloning and sequencing of the variable regions of these two MAb indicated that both heavy chain variable regions were originated from the usage of the same germinal V and J genes. However, the outstanding differences in the size of the VH CDR3, and the absolute difference in the light chain sequences, suggest that the hybridomas were originated from different precursor B lymphocytes. With respect to their use as immunoaffinity chromatography ligands for the purification of a recombinant HBsAg, we found that the IgM immunogel exhibited increased performance with respect to amount of eluted antigen, and final recovery. This difference in overall performance could be attributed to a series of factors: the higher valence number of IgM, a dissimilar distribution of IgM and IgG in the activated gel particles, and differences in antigen recognition between both MAb. Our results suggest that IgM antibodies may be useful in immunopurification, particularly if the antigen is structurally complex and has a high density of repeating epitopes.

Amino Acid Sequence↗
PubMed1996

Epitope mapping, V-region DNA sequence, and neutralizing Fab fragments of two monoclonal antibodies against the HIV-1 V3 loop.

BACKGROUND: Experimental evidence suggests that neutralizing antibodies could constitute an important factor in the control of AIDS progression and that the V3 loop of gp120 constitutes the main target for such purposes. We have previously developed two neutralizing murine monoclonal antibodies (Mabs) against the V3 region of the HIV-1 MN strain. OBJECTIVES: To characterize those Mabs in terms of fine specificity and DNA sequence of their V regions and to study if Fab fragments retain their neutralizing potential in vitro. STUDY DESIGN: A set of 12-mer alanine substituted peptides were employed for epitope mapping using two ELISA procedures: (1) indirect, with each peptide bound to polystyrene plates, and (2) competitive, with co-incubation of peptides and Mabs in solution. The V regions of both Mabs were PCR amplified from cDNA and their nucleotide sequences were determined. Finally, Fab fragments of Mab 10F10 were generated and their neutralizing capacity against the MN isolated was assessed. RESULTS: We first restricted the minimal length of the epitopes recognized by 2C4 and 10F10 to the 12-mer peptide KRIHIGPGRAFY. The core of the epitopes recognized by Mabs 2C4 and 10F10 were IHIGP-R and IHIG-R, respectively. While substitution of proline in position 7 completely abolished the binding of 2C4, it only reduced that of 10F10 by 50%. Finally, Fab fragments of Mab 10F10 were still able to neutralize the HIV-1 MN strain in vitro. CONCLUSION: This subtle distinction in the fine mapping of the epitope recognized by Mabs 2C4 and 10F10 should correspond to three amino acid differences that we found in the heavy chain V-regions. The Fab fragments of Mab 10F10 retained the neutralizing capacity. This indicates that HIV neutralization by anti V3 Mabs is an Fc independent process.

Amino Acid Sequence↗
PubMed1996

Production, purification and characterization of an anti-(carcinoembryonic antigen) recombinant single-chain Fv antibody fragment.

Specific targeting of radioactive agents to tumour cells has been the main objective of the in vivo use of monoclonal antibodies and their fragments. In particular, specific antibodies to carcinoembryonic antigen (CEA)-expressing tumours can be used either for diagnosis or therapy, if targeting could be improved. The expression of antibody fragments in bacteria allows the preparation of engineered molecules with antigen-binding properties and a better penetration into the tumour. A specific anti-CEA single-chain Fv fragment was produced in bacteria and purified. Its binding activity has been demonstrated in ELISA, immunocytochemistry, immunohistochemistry, fluorescence-activated cell sorting and the kinetic parameters determined by the plasmon surface resonance.

Antibodies, Neoplasm↗
PubMed1996

Vitamin D prophylaxis in children with a single dose of 150000 IU of vitamin D.

OBJECTIVE: To determine the efficacy of a single oral dose of 150,000 IU of vitamin D2 at the beginning of autumn for preventing winter vitamin D deficiency in children in Ushuaia (55 degrees S). DESIGN: The study was prospective. SUBJECTS: 79 children clinically healthy with 8.6 +/- 1.4 y of age (X +/- s.d.). INTERVENTIONS: Fasting serum venous samples and 2 h urine samples were obtained immediately before and 6 w and 5 mon after the vitamin D dose. Parents informed consent was obtained previous to the study. In a subgroup of 30 children serum levels of calcium (sCa), phosphorus (sP), total alkaline phosphatase (TAP), 25 hydroxyvitamin D (25 OHD), parathyroid hormone (PTH) and the urine calcium/creatinine ratio in a 2 h urine sample (UCa/UCreat) were measured. In the whole group sCa and the ratio uCa/ucreat were measured. RESULTS: After 150,000 IU of vitamin D2 administration, serum 25 OHD levels at the end of winter (17.0 +/- 9.4 ng/ml) were similar to those at the beginning of autumn (18.7 +/- 10.7 ng/ml), but significantly higher from those obtained in a previous study without vitamin D (9.8 +/- 3.8 ng/ml, P < 0.001). PTH levels were higher at the end of winter (P < 0.02), but this augmentation was lower than the increment observed without vitamin D. Plasma calcium levels and the urine calcium/creatinine ratio were lower at 5 months after vitamin D2 dose (P < 0.02 and P < 0.05 respectively). In the total group the serum calcium was lower after the fifth month (P < 0.05). The Uca/Ucreat ratio was lower at 6 w and 5 mon (P < 0.05 and P < 0.001). CONCLUSION: A single dose of 150,000 IU of vitamin D maintained appropriate levels of 25 OHD without inducing hypercalcemia nor hypercalciuria, but a winter increment of PTH (smaller than in the group without vitamin D) was not inhibited.

Alkaline Phosphatase↗
PubMed1995

A point mutation in a murine immunoglobulin V-region strongly influences the antibody yield in Escherichia coli.

Recombinant DNA technology has made it possible to produce specific Fab and scFv antibody (Ab) fragments in prokaryotic host cells. Using vectors designed for periplasmic expression of encoded Ab fragments, we have been studying how the sequence and genetic localization of the light chain (L-chain) variable region gene of a mouse Ab (CB-Nm.1) determined the level of Ab production. The variable region was shown to belong to the V kappa V family and contained a previously unreported Ile72. Nine different Ab constructions were tested in monocistronic (scFv) or dicistronic (Fab) operons for their ability to affect the synthesis level of the L-chain. When the gene coding for the L-chain was located downstream from the Fd fragment gene, the substitution of codons encoding Ile by a codon encoding Thr was found to be crucial for any expression of the L-chain fragment. This was, however, not accompanied by an increase in L-chain-specific mRNA, neither was there any change in the size of the mRNA. The fact that the unmutated L-chain protein was produced from cells transformed with certain other constructions indicated that the protein as such was not incompatible with the prokaryotic environment. Together, this suggested that the translation process was involved in the restricted production of the L-chain. Thus, surprisingly small substitutions significantly affected the expression level, a fact that will have important implications on the library size expressed in prokaryotic hosts, including phage-displayed Ab libraries.

Base Sequence↗
PubMed1995

Variable region sequence modulates periplasmic export of a single-chain Fv antibody fragment in Escherichia coli.

Using PCR, we have cloned antibody heavy and light chain variable region (VH and VL) coding sequences specific for a recombinant hepatitis B virus surface antigen (HBsAg) and assembled these for expression as single-chain Fv (scFv) fragments in Escherichia coli periplasm using the ompA signal peptide. The vectors also encoded N- or C-terminal His6 extensions to allow for the purification of the expressed proteins using immobilized metal affinity chromatography (IMAC). We found that the VH-linker-VL configuration of the scFv was not exported to the periplasm but remained associated with cellular insoluble material, from which it could be extracted, renatured to its active form by gentle dialysis and purified using IMAC. The molecular size of the scFv suggests that the ompA signal peptide was not processed. Based on previous reports, we hypothesized that the arginine in framework 1 (FR1) of the VH might interfere with translocation to the periplasm by means of the signal peptide. Because no arginines are present in FR1 of VL, we reversed the order of the V-regions in the scFv and observed efficient export of the active scFv to the periplasm. Furthermore, when the arginine in FR1 of VH was mutated to glycine in the original VH-linker-VL construct, active scFv was also exported to the periplasm. Thus, exposed positive charges near the signal peptide may account for at least some of the often-encountered difficulties in bacterial scFv expression.

Amino Acid Sequence↗
PubMed1995

Purification and application of a single-chain Fv antibody fragment specific to hepatitis B virus surface antigen.

Immobilized metal affinity chromatography (IMAC) has been recently applied to the purification of of recombinant proteins bearing multi-histidine domains at their N or C terminus. We have now used this procedure for the single-step purification of an anti-Hepatitis B virus surface antigen (HBsAg) single-chain Fv (scFv) antibody fragment. Adjusting the metal ion (Cu+2 or Ni+2) and elution conditions (pH or imidazole), we efficiently separated active scFv forms from inactive molecules. Achieved purity was 93%, with a 20% yield with respect to the scFv content in the initial material. The pure scFv was coupled to CNBr-activated Sepharose 4B and compared the original monoclonal antibody (MAb) CB-Hep.1 in the immunoaffinity purification of a vaccine recombinant HBsAg (r-HBsAg). Results indicate that eluted antigen per mg of coupled ligand was similar for the scFv and the MAb when pure r-HBsAg was used as starting material. Preliminary results with unpurified starting material are also encouraging.

Antibodies, Monoclonal↗
PubMed1994

Intra- and extracellular expression of an scFv antibody fragment in E. coli: effect of bacterial strains and pathway engineering using GroES/L chaperonins.

We have studied the influence of bacterial host on the secretion of single-chain Fv antibody fragment (scFv), the production of this antibody fragment as intracellular fusion protein, and the effect of chaperonin coexpression on intracellular antibody expression. Seven bacterial strains were transformed with a vector carrying the genes encoding the variable regions of an anti-CEA scFv antibody and the ompA leader sequence (ptrp/ompA/scFvCEA). Expression and secretion of this antibody fragment were highest in the W3110 strain, as determined by Western blot analysis and enzyme immunoassay, where the scFv fragment amounted to approximately 30% of the total periplasmic protein. Except for BMH71-18, the other strains were unsuitable for antibody fragment expression, suggesting screening of bacterial strains as an important parameter. For intracellular expression, the scFv was expressed as a fusion protein with a 26-amino acid N-terminal fragment of human interleukin-2 (IL-2), using the pIL-2f/scFvCEA vector. The fusion protein was expressed at 30% of total biomass and retained antigen binding after in vitro refolding. Co-expression of chaperonin-encoding plasmid pGroES/L with pIL-2f/scFv increased the intracellular production of the fusion protein twofold, with a similar increase in the final amount of active scFv antibody fragment that could be obtained after in vitro refolding. The chaperonins had no effect on secretion of scFv antibody fragments, using the ptrp/ompA/scFvCEA.

Bacterial Proteins↗
PubMed1993

Chaperonin assisted phage display of antibody fragments on filamentous bacteriophages.

We have used the GroE chaperonins to assist in the packing of a new phage display vector, pEXmide3. Titers of the packed phagemid increased almost 200-fold from approximately 4 x 10(11) cfu/ml, without coexpression of the GroE proteins, to approximately 7 x 10(13) cfu/ml with their coexpression. Equal titers of non-assisted and assisted phagestocks exhibited the same antigen specificity and ELISA reactivity, indicating the same frequency of displayed Fab-fragments. While the diversity of antibody libraries depends on the bacterial transformation efficiency, the copy number of each antibody is determined by subsequent amplification of the phage, thus chaperonin assisted phagemid packing in bacteriophage M13 can be used as a general and simple tool to increase the amplification level of expressed Fab fragments. pEXmide3 was developed for display of Fab and single chain Fv-fragments (scFv), using restriction enzymes that do not cut, or cut with low frequencies, in genes encoding immunoglobulin variable domains. The vector allows cloning of genes for the variable domains linking these to predetermined human constant domains or cloning of the entire light and heavy Fab chains. A modification of the pelB leader sequence, with a glutamine to alanine substitution at residue 18, was used for export of the light chain.

Bacterial Proteins↗