Antioxidant defense in the follicular fluid of water buffalo.

Oxidative damages to the oocyte or follicular cells were suggested to trigger atresia. In water buffalo, loss of the blood-follicle barrier sieving effect on the diffusion of plasma haptoglobin was previously found to be associated with atretic oocytes. The redox status of water buffalo follicles was evaluated by measuring in follicular fluid both the total antioxidant capacity (TAC), expressed as Trolox equivalents, and the concentration of specific free radical scavengers, determined by high-performance liquid chromatography. Among follicles at random stages of the reproductive cycle (n = 74), a number (n = 32) were analyzed also for the cumulus-oocyte morphology or plasma haptoglobin penetration. The haptoglobin follicular concentration compatible with the barrier selectivity function was calculated to be less than 53% of the concentration in plasma. The data on TAC, retinol, alpha-tocopherol, gamma-tocopherol, ascorbic acid, and uric acid fluctuated in a wide range. The relative (follicular vs. plasmatic) levels of alpha-tocopherol were found to be negatively correlated with those of retinol (p < 0.01). In the follicles, the alpha-tocopherol levels were 1.25 +/- 0.35 or 1.99 +/- 0.72 microM when the haptoglobin concentration was <53 or >53% of the concentration in plasma, respectively. The concentration of ascorbic acid or uric acid was higher (up to 10- or 30-fold, respectively) in follicular fluid than in plasma. Fluids containing haptoglobin >53% or associated with cumulus-oocyte complexes of bad quality displayed levels of uric acid about 20-fold higher than in plasma. The results suggest that a high penetration of haptoglobin in the follicle and cumulus-oocyte degradation is associated with alterations of the level of the major antioxidants, particularly with enhancement of the uric acid concentration.

Human Rad50 is physically associated with human Mre11: identification of a conserved multiprotein complex implicated in recombinational DNA repair.

In this report, we describe the identification and molecular characterization of a human RAD50 homolog, hRAD50. hRAD50 was included in a collection of cDNAs which were isolated by a direct cDNA selection strategy focused on the chromosomal interval spanning 5q23 to 5q31. Alterations of the 5q23-q31 interval are frequently observed in myelodysplasia and myeloid leukemia. This strategy was thus undertaken to create a detailed genetic map of that region. Saccharomyces cerevisiae RAD50 (ScRAD50) is one of three yeast RAD52 epistasis group members (ScRAD50, ScMRE11, and ScXRS2) in which mutations eliminate meiotic recombination but confer a hyperrecombinational phenotype in mitotic cells. The yeast Rad50, Mre11, and Xrs2 proteins appear to act in a multiprotein complex, consistent with the observation that the corresponding mutants confer essentially identical phenotypes. In this report, we demonstrate that the human Rad50 and Mre11 proteins are stably associated in a protein complex which may include three other proteins. hRAD50 is expressed in all tissues examined, but mRNA levels are significantly higher in the testis. Other human RAD52 epistasis group homologs exhibit this expression pattern, suggesting the involvement of human RAD52 epistasis group proteins in meiotic recombination. Human RAD52 epistasis group proteins are highly conserved and act in protein complexes that are analogous to those of their yeast counterparts. These findings indicate that the function of the RAD52 epistasis group is conserved in human cells.

Complementation of the radiosensitive phenotype in severe combined immunodeficient mice by human chromosome 8.

Severe combined immunodeficient (scid) C.B-17 mice are deficient in variable (diversity) joining region recombination, the process of assembling the immunoglobulin and T-cell receptor genes from gene segments, thereby creating much of the enormous diversity of antigen-binding capacity, scid mice are also sensitive to ionizing radiation, as a result of their deficiency in double-strand break repair. Here we report the complementation of the radiation-sensitive scid phenotype by transferring human chromosome 8 into scid cells. Somatic cell hybrids were generated by fusing scid cells with human HT-1080 cells, resulting in radioresistant hybrids with several human chromosomes. One of the identified human chromosomes in the radioresistant scid cell line 4.61, which retains only two human chromosomes, is a rearranged 8/21 translocation. Proof that chromosome 8 confers the complementation was achieved by transferring only human chromosome 8 into scid cells by microcell-mediated chromosome transfer (scid/hu8 cell line). The presence of chromosome 8 in our scid/hu8 cell line was monitored by fluorescence in situ hybridization and polymerase chain reaction. We demonstrated the radioresistance of this hybrid not only to high dose rate but also to low dose rate radiation. We also showed that transference of human chromosome 8 to scid cells fully complements the DNA double-strand break repair deficiency and the high sensitivity of scid cells to radiation-induced chromosome aberrations. Mapping the scid gene to human chromosome 8 is an important first step in cloning the scid gene, which will enhance our understanding of double-strand break repair pathways in humans.

Characterization of a CHO cell line resistant to killing by the hypoxic cell cytotoxin SR 4233.

One approach to understanding the mechanism of selective hypoxic cell killing by the benzotriazine-di-N-oxide, SR 4233, is to characterize cell lines that exhibit increased resistance to killing by this drug. The Chinese Hamster Ovary cell line BL-10 was originally isolated on the basis of its hypersensitivity to killing by bleomycin. It is 2.7-fold more resistant to hypoxic cell killing by SR 4233 than wild-type CHO on comparison of D0's. However, both BL-10 and CHO possess the same sensitivity to killing by SR 4233 under aerobic conditions. We have excluded the explanation that differential metabolism of SR 4233 is responsible for its increased survival as both BL-10 and CHO produce the two-electron product SR 4317 at the same rate (3 nmoles/hr/10(6) cells). Analysis of free radical production, DNA double-strand break induction, and glutathione (GSH) levels suggested that the resistance of BL-10 to killing by SR 4233 might result from increased intracellular radical scavenger pathways. Using buthionine sulfoximine (BSO) to decrease cellular GSH levels, we found a marked increase in the sensitivity of BL-10 cells to SR 4233 killing under hypoxia, but a much smaller increase in the sensitivity of CHO cells. Taken together, these data imply that the high GSH levels in BL-10 cells is responsible for its resistance to SR 4233 cytotoxicity.

Second-generation 1,2,4-benzotriazine 1,4-di-N-oxide bioreductive anti-tumor agents: pharmacology and activity in vitro and in vivo.

SR 4233 (1,2,4-benzotriazine-3-amine 1,4-dioxide) will soon be entering Phase I clinical trials as a new bioreductive cytotoxic agent for the treatment of solid tumors in combination with fractionated radiotherapy. We have selected 3 from over 50 analogues of SR 4233 which showed particular promise as second generation bioreductive antitumor agents. These compounds, when compared to SR 4233, have higher hypoxic toxicity and comparable or higher oxic to hypoxic cytotoxicity ratios in vitro and similar animal toxicity. We have compared the effectiveness of these three compounds with SR 4233 in two tumor systems and have examined some pharmacokinetic properties. The results show that replacement of the amino group at the 3-position of SR 4233 with either a hydrogen or an N,N-dialkylaminoalkylamino group shortens the half-life of these compounds in the blood because of the combined effects of partition coefficients, basicity, and higher reactivity. SR 4754 and SR 4755, the N,N-dialkylaminoalkylamino derivatives, exhibited shorter plasma half-lives than SR 4233 but exhibited lower anti-tumor activity than SR 4233 based on equal mouse toxicity in a fractionated regimen. SR 4482, with the hydrogen substitution and very high electron affinity, possessed a very short blood half life yet retained similar anti-tumor activity as SR 4233.

scid mutation in mice confers hypersensitivity to ionizing radiation and a deficiency in DNA double-strand break repair.

C.B-17 severe combined immunodeficient (scid) mice carry the scid mutation and are severely deficient in both T cell- and B cell-mediated immunity, apparently as a result of defective V(D)J joining of the immunoglobulin and T-cell receptor gene elements. In the present studies, we have defined the tissue, cellular, and molecular basis of another characteristic of these mice: their hypersensitivity to ionizing radiation. Bone marrow stem cells, intestinal crypt cells, and epithelial skin cells from scid mice are 2- to 3-fold more sensitive when irradiated in situ than are congenic BALB/c or C.B-17 controls. Two independently isolated embryo fibroblastic scid mouse cell lines display similar hypersensitivities to gamma-rays. In addition, these cell lines are sensitive to cell killing by bleomycin, which also produces DNA strand breaks, but not by the DNA crosslinking agent mitomycin C or UV irradiation. Measurement of the rejoining of gamma-ray-induced DNA double-strand breaks by pulsed-field gel electrophoresis indicates that these animals are defective in this repair system. This suggests that the gamma-ray sensitivity of the scid mouse fibroblasts could be the result of reduced repair of DNA double-strand breaks. Therefore, a common factor may participate in both the repair of DNA double-strand breaks as well as V(D)J rejoining during lymphocyte development. This murine autosomal recessive mutation should prove extremely useful in fundamental studies of radiation-induced DNA damage and repair.

The efficacy, safety and tolerance of ceftazidime for the treatment of bacterial infections in the elderly.

An open multicentre trial to study the efficacy and safety of ceftazidime in elderly patients has been conducted in four geriatric units on 135 subjects suffering from urinary-tract or respiratory-tract infections. Sixty-two patients were cured (45.9%), and 60 improved (44.4%). Of the evaluable cases bacteriological eradication was achieved in 91.3%. No adverse events were recorded.

[Syphilitic pleuritis. Description of a clinical case]. / Pleurite luetica. Descrizione di un caso clinico.

A rare case of luetic pleurisy diagnosed in a patient with tertiary syphilis, when the aetiological agent was discovered in the pleuritic exudate is described. The spirochaetes, first revealed by dark field microscopy, were studied further under the electron microscope, using negative colouring and fine sections.