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Biomedical subjects

R Lorenzini

Publications and source records attributed to R Lorenzini.

At least 19 recordsLinked to original sources

Active-feedback control of the magnetic boundary for magnetohydrodynamic stabilization of a fusion plasma.

Stable operation with control on magnetohydrodynamic modes has been obtained in the modified reversed field experiment employing a set of 192 feedback controlled saddle coils. Improvements of plasma temperature, confinement (twofold), and pulse length (threefold) and, as a consequence of the magnetic fluctuation reduction, strong mitigation of plasma-wall interaction and mode locking are reported.

Journal Article↗

Species identification and confirmation of human and animal cell lines: a PCR-based method.

Misidentification and cross-contamination of cell lines are major problems of cell cultures that can make scientific results and their reproducibility unreliable. This paper describes a PCR-based method for easily identifying or confirming the species of origin of cell lines by using a panel of oligonucleotides specific for the nine animal species most common in cell culture laboratories. A panel of 35 human and animal cell lines, whose species of origin were previously confirmed by isoenzyme assay, was studied with nine species-specific primer pairs that specifically anneal to DNA sequences codifying for human, cat, dog, mouse, rat, horse, rabbit, African Green monkey cytochrome c oxidase subunit I (cox I), and one primer pair specific for the cytochrome b gene of Chinese hamster. The amplified fragments were analyzed by electrophoresis in ethidium bromide-stained 2% agarose gels. The method is simple, rapid, highly sensitive, and useful for routinely monitoring the species identity of cell cultures.

Animals↗

Accentuated response to phenylhydrazine and erythropoietin in mice genetically impaired for their GATA-1 expression (GATA-1(low) mice).

The response of mice genetically unable to up-regulate GATA-1 expression (GATA-1(low) mice) to acute (phenylhydrazine [PHZ]-induced anemia) and chronic (in vivo treatment for 5 days with 10 U erythropoietin [EPO] per mouse) erythroid stimuli was investigated. Adult GATA-1(low) mice are profoundly thrombocytopenic (platelet counts [x 10(9)/L] 82.0 +/- 28.0 vs 840 +/- 170.0 of their control littermates, P <.001) but have a normal hematocrit (Hct) (approximately.47 proportion of 1.0 [47%]). The spleens of these mutants are 2.5-fold larger than normal and contain 5-fold more megakaryocytic (4A5(+)), erythroid (TER-119(+)), and bipotent (erythroid/megakaryocytic, TER-119(+)/4A5(+)) precursor cells. Both the marrow and the spleen of these animals contain higher frequencies of burst-forming units-erythroid (BFU-E)- and colony-forming units-erythroid (CFU-E)-derived colonies (2-fold and 6-fold, respectively) than their normal littermates. The GATA-1(low) mice recover 2 days faster from the PHZ-induced anemia than their normal littermates (P <.01). In response to EPO, the Hct of the GATA-1(low) mice raised to.68 proportion of 1.0 (68%) vs the.55 proportion of 1.0 (55%) reached by the controls (P <.01). Both the GATA-1(low) and the normal mice respond to PHZ and EPO with similar (2- to 3-fold) increases in size and cellularity of the spleen (increases are limited mostly to cells, both progenitor and precursor, of the erythroid lineage). However, in spite of the similar relative cellular increases, the increases of all these cell populations are significantly higher, in absolute cell numbers, in the mutant than in the wild-type mice. In conclusion, the GATA-1(low) mutation increases the magnitude of the response to erythroid stimuli as a consequence of the expansion of the erythroid progenitor cells in their spleen.

Anemia↗

Normal and cancer-prone human cells respond differently to extremely low frequency magnetic fields.

Human lymphoblastoid cells of normal origin and from genetic instability syndromes, i.e. Fanconi anemia (FA) group C and ataxia telangectasia, were continuously exposed to extremely low frequency magnetic field (ELF-MF). We report that ELF-MF, though not perturbing cell cycle progression, increases the rate of cell death in normal cell lines. In contrast, cell death is not affected in cells from genetic instability syndromes; this reflects a specific failure of the apoptotic response. Reintroduction of complementation group C in FA cells re-established the apoptotic response to ELF-MF. Thus, genes implicated in genetic instability syndromes are relevant in modulating the response of cells to ELF-MF.

Apoptosis↗

Specific localization of transcription factors in the chromatin of mouse mature spermatozoa.

We previously characterized a nuclease-hypersensitive fraction of mouse sperm chromatin, which is organized in a typical nucleosomal structure. A partial genomic library was constructed with the DNA from the nuclease-hypersensitive chromatin, which revealed a high content in retroposon/retroviral DNA sequences. Here we report that the cloned nuclease-hypersensitive DNA also contains clusters of potential sites for transcription factors: among those, binding sites for Oct-1, Oct-4, TBP, Ets-1, and C/EBP are most abundant. This observation prompted us to ask whether mature spermatozoa contain the corresponding protein factors. Indirect immunofluorescence experiments show that all analyzed factors are indeed present in the sperm heads. Moreover, transcription factors are associated with the nuclease-hypersensitive chromatin of spermatozoa, as endogenous nucleases that degrade the hypersensitive fraction also cause the concomitant release of transcription factors from sperm cells into the medium. Band-shift assays with proteins extracted from the supernatant, and immunofluorescence analysis of sperm pellets, indicate that transcription factors are largely recovered in the supernatant while being absent or poorly retained in spermatozoa. The possible involvement of these factors in early embryogenesis is discussed.

Animals↗

Reverse transcriptase activity in mature spermatozoa of mouse.

We show here that a reverse transcriptase (RT) activity is present in murine epididymal spermatozoa. Sperm cells incubated with human poliovirus RNA can take up exogenous RNA molecules and internalize them in nuclei. Direct PCR amplification of DNA extracted from RNA-incubated spermatozoa indicate that poliovirus RNA is reverse-transcribed in cDNA fragments. PCR analysis of two-cell embryos shows that poliovirus RNA-challenged spermatozoa transfer retrotranscribed cDNA molecules into eggs during in vitro fertilization. Finally, RT molecules can be visualized on sperm nuclear scaffolds by immunogold electron microscopy. These results, therefore, reveal a novel metabolic function in spermatozoa, which may play a role during early embryonic development.

Animals↗

Nucleosomal domains of mouse spermatozoa chromatin as potential sites for retroposition and foreign DNA integration.

Exogenous DNA molecules are spontaneously taken up by sperm cells, internalized in nuclei, and eventually integrated in the sperm genome. The actual occurrence of the integration suggests that the sperm chromosomal DNA is not uniformly and tightly packed with protamines, implying the existence of genomic sites where the chromosomal DNA is accessible to foreign molecules. We have characterized a hypersensitive, nucleosomal subfraction of mouse sperm chromatin that is highly enriched in unmethylated retroposon DNA from a variety of families. Here we propose that both the integration of exogenous DNA molecules, and the endogenous retroposition activity, occur in the same site(s) of sperm chromatin.

Animals↗

DNA dose and sequence dependence in sperm-mediated gene transfer.

We have tested three parameters in sperm-mediated gene transfer assays with mice and pigs: (i) the epididymal versus ejaculated origin of sperm cells, (ii) the primary structure, and (iii) the amount of the challenging foreign DNA. We have found that the pVLCNhGH construct, of retrotransposon origin, causes a massive embryo lethality and yet increases the yield of genetic transformation among born animals of both species compared to viral constructs. Arrest of embryonic development is a DNA dose-dependent effect, which is observed with high DNA doses, while lower doses are compatible with development. Finally, the overall efficiency of sperm-mediated gene transfer is higher when ejaculated, versus epididymal, spermatozoa are used. We suggest that this difference is related to the highly efficient apoptotic response in epididymal compared to ejaculated spermatozoa, triggered by the interaction of exogenous DNA molecules with the sperm membrane.

Animals↗

Matrix solid-phase dispersion microextraction and determination by high-performance liquid chromatography with UV detection of pesticide residues in citrus fruit.

A multiresidue method based on matrix solid-phase dispersion (MSPD) microextraction was studied to determine the carbamate, benfuracarb, and urea insecticides, diflubenzuron, flufenoxuron hexaflumuron and hexythiazox, used in control of citrus pests. Optimisation of different parameters, such as the type of solid support for matrix dispersion, elution solvents and the clean-up step were carried out. The method used 0.5 g of orange sample, C8 bonded silica as MSPD sorbent and dichloromethane as eluting solvent. Recoveries, at spiked concentrations below the maximum residue levels established by Spanish Government, were between 74 and 84% with relative standard deviations ranging from 2 to 4%. The limits of quantification were from 0.15 to 0.25 microgram/g using high-performance liquid chromatography with UV detection at 200 nm. The method may be useful as a screening protocol for the determination of these newly developed pesticides in citrus samples.

Benzamides↗

A fraction of mouse sperm chromatin is organized in nucleosomal hypersensitive domains enriched in retroposon DNA.

We have characterized a nuclease hypersensitive chromatin fraction from murine spermatozoa. Endogenous nuclease activity can be induced in mouse epididymal spermatozoa by appropriate stimuli and cause the localized degradation of chromosomal DNA. Based on these observations, we have isolated nuclease hypersensitive chromatin regions released from spermatozoa in the supernatant of pelleted sperm cells, and have cloned and characterized the DNA. Gel electrophoresis of end-labelled released DNA fragments showed a typical nucleosomal distribution. Peripherally distributed nucleohistones were visualized by immunofluorescence in sperm nuclei, and histones were identified by western blot in sperm chromatin. Moreover, the released DNA is enriched in retroposon DNA from a variety of families. FISH and immunofluorescence analysis showed that retroposon DNA and nucleohistone chromatin co-localize and are both peripherically distributed in nuclei of spermatozoa. In contrast, a major satellite DNA probe, used for control, co-localizes with highly condensed chromatin in the central region of sperm nuclei. The nuclear Ran and RCC1 proteins were also visualized in the dorsal margin of sperm nuclei, and were abundantly released with the hypersensitive chromatin fraction. Together, these results indicate that nucleohistone chromatin fraction(s) with typical features of 'active' chromatin are present in murine spermatozoa, are hypersensitive to nuclease cleavage, enriched in retroposon DNA and organized in nucleosomal domains. These observations suggest that nucleohistone domains identify a fraction of the sperm genome which may be functional during early embryogenesis.

Animals↗

Increased production of mouse embryos in in vitro fertilization by preincubating sperm cells with the nuclease inhibitor aurintricarboxylic acid.

Exposure of spermatozoa to stress conditions causes a drastic reduction of their fertilizing ability. We report here that the decrease in fertilization can be effectively antagonized by preincubating sperm cells with the nuclease inhibitor drug aurintricarboxylic acid (ATA). Preincubation of mouse epididymal sperm cells with ATA increased the yield of 2-cell embryos produced by in vitro fertilization assays. The effect of ATA was selectively exerted via spermatozoa, since neither preincubation of eggs, nor the direct treatment of zygotes, modified the yield of 2-cell-stage embryos. Our results suggest that ATA does not directly improve the ability of sperm cells to penetrate the egg cytoplasm but instead acts by preserving sperm nuclei from induced or spontaneously occurring damage and/or favors events that trigger early embryogenesis.

Animals↗

Bcl-2 and Bax expression in thyroid tumours. An immunohistochemical and western blot analysis.

Bcl-2 and Bax proteins, which are involved in repressing and promoting programmed cell death, respectively, have been investigated immunohistochemically and by Western blot analysis in a series of thyroid tumours. Three immunostaining patterns were identified. Benign lesions and well-differentiated thyroid carcinomas displayed a profile similar to that of normal follicular epithelium, in which Bcl-2 immunostaining was predominant. Thyroid carcinomas associated with an aggressive behaviour, such as the tall-cell variant of papillary carcinoma and the poorly differentiated carcinomas, co-expressed both proteins. Finally, anaplastic carcinomas expressed only the Bax protein. Western blot analyses revealed that the anti-Bcl-2 antibody recognized two bands, of molecular weights 21 kDa and 25 kDa. This was only seen in the tall-cell papillary carcinomas and in the anaplastic carcinomas.

Adult↗

Activation of endogenous nucleases in mature sperm cells upon interaction with exogenous DNA.

Mature sperm cells, either of epididymal origin or ejaculated and depleted of seminal fluid, are spontaneously able to bind exogenous DNA molecules which are subsequently internalized into sperm nuclei. Southern blot analysis showed that the internalized DNA was specifically cleaved by sperm endonucleases and showed typical fragmentation patterns of localized hypersensitivity. Nucleases were activated in response to the internalization of exogenous DNA by sperm cells and their activity increased with the DNA concentration. Nuclease activation was efficient in epididymal sperm cells, while being drastically reduced in ejaculated washed spermatozoa. Nucleases were Ca++ dependent, and were, respectively, inhibited and activated by preincubating sperm cells with Aurintricarboxylic Acid (ATA) and Ca++ Ionophore A23187, which are known to, respectively, inhibit and activate apoptosis in somatic cells. Moreover, nuclease activation also caused a partial degradation of the sperm endogenous chromosomal DNA; cleaved DNA fragments were released from the sperm cells to the medium. Taken together, these results suggest that a metabolically active process similar to apoptosis is triggered in the nuclei of mature sperm cells upon interaction with exogenous DNA.

Animals↗

Idiotypic vaccination: immunoprotection mediated by anti-idiotypic antibodies with antibiotic activity.

Anti-Id antibodies were raised in mice against a monoclonal antibody (MoAb KT4) that neutralized the in vitro activity of a Pichia anomala yeast killer toxin. Monoclonal antibody was administered to BALB/C syngeneic mice with different schedules of immunization before intravenous challenge with increasing amounts of yeast killer toxin-sensitive Candida albicans cells. The course of candidosis was studied in comparison with mice non-immunized and immunized with an isotype-matched unrelated MoAb subdivided into control groups. Protection was reflected by statistically significant increases in survival rate of mice immunized with MoAb KT4 which showed variable serum levels of yeast killer toxin-like anti-Id antibodies. MoAb KT4 affinity chromatography purified mouse anti-Id antibodies were capable of killing in vitro the yeast cells of the Candida albicans strain used for the experimental infection. This is the first report of antimicrobial protection that exploits the role of anti-idiotypic antibodies presumably acting in vivo as antibiotics (idiotypic vaccination).

Animals↗

Filamentous growth and elevated vaginopathic potential of a nongerminative variant of Candida albicans expressing low virulence in systemic infection.

The vaginopathic potential and the intravaginal morphology of a nongerminative variant of Candida albicans, strain CA-2, were studied in a rat vaginitis model. Although it expressed low virulence in systemic infections, strain CA-2 was capable of causing a vaginal infection of the same duration and extent as that obtained in rats challenged with the germ-tube-forming strain C. albicans 3153 from the stock collection or with a fresh clinical isolate of C. albicans from a case of human vaginitis. During the experimental infection, the CA-2 cells did not maintain their yeast morphology but gave rise to single enlarged-elongated elements (1 to 2 days) which grew predominantly as coarse, short, pseudomycelium-like filaments (2 to 3 days) and then as long threads (7 days). These latter filaments were ultimately indistinguishable from the hyphal filaments formed by the germ-tube-forming strains, which, however, initially developed in the vagina by typical germ tube formation. This peculiar morphological development of strain CA-2 was not observed in organs of systemically infected mice, where, in contrast to strain 3153 which formed typical hyphae, strain CA-2 maintained a typical pattern of yeast growth. Vaginal isolates of strain CA-2 taken at different days of infection were found to be identical to the challenging CA-2 cells, in terms of biochemical characteristics, inability to form germ tubes in any medium at 37 degrees C in vitro, echinocandin resistance, DNA biotype, and low virulence in systemic infections in mice. Thus, experimental vaginitis by strain CA-2 is associated with a peculiar filamentous growth in the vagina, through an apparently novel morphological development bypassing classical germ tube formation but ultimately leading to ordinary hyphae. The elevated vaginopathic potential of strain CA-2, in contrast to its low virulence in systemic infection, also suggests that different Candida virulence factors (and host responses) come into play in local and disseminated candidal infections.

Acute Disease↗

[Intra- and postoperative anesthesiologic management in cerebral arteriovenous malformations].

The authors analyze the anesthetic management of 59 cases of "large" arteriovenous malformation (more than 20 cm3), all completely removed by microsurgical technique. The discussion is focused on immediate pre-operative, intra-operative and post-operative pharmacological treatments in order to reduce the hemodynamic effects in the surrounding brain after excision of angiomas.

Anesthesia↗

Application of a high-performance liquid chromatography coulometric method for the estimation of mebendazole and its metabolites in human sera.

A novel, sensitive high-performance liquid chromatographic method, making use of coulometric detection, for the estimation of mebendazole and its metabolites in the sera of eight hydatidosis patients was devised. Recovery rates, precision, accuracy and sensitivity for each compound are reported and compared with those of the previously published methods.

Biotransformation↗