Oral Administration of Electron-Beam Inactivated Rhodococcus equi Failed to Protect Foals against Intrabronchial Infection with Live,Virulent R. equi

There is currently no licensed vaccine that protects foals against Rhodococcus equi–induced pneumonia. Oral administration of live, virulent R. equi to neonatal foals has been demonstrated to protect against subsequent intrabronchial challenge with virulent R. equi. Electron beam (eBeam)-inactivated R. equi are structurally intact and have been demonstrated to be immunogenic when administered orally to neonatal foals. Thus, we investigated whether eBeam inactivated R. equi could protect foals against developing pneumonia after experimental infection with live, virulent R. equi. Foals (n = 8) were vaccinated by gavaging with eBeam-inactivated R. equi at ages 2, 7, and 14 days, or gavaged with equal volume of saline solution (n = 4), and subsequently infected intrabronchially with live, virulent R. equi at age 21 days. The proportion of vaccinated foals that developed pneumonia following challenge was similar among the vaccinated (7/8; 88%) and unvaccinated foals (3/4; 75%). This vaccination regimen did not appear to be strongly immunogenic in foals. Alternative dosing regimens or routes of administration need further investigation and may prove to be immunogenic and protective.     

Y chromosome microdeletions in Brazilian fertility clinic patients

Microdeletions in Yq are associated with defects in spermatogenesis, while those in the AZF region are considered critical for germ cell development. We examined microdeletions in the Y chromosomes of patients attended at the Laboratory of Human Reproduction of the Clinical Hospital of the Federal University of Goiás as part of a screening of patients who plan to undergo assisted reproduction. Analysis was made of the AZF region of the Y chromosome in men who had altered spermograms to detect possible microdeletions in Yq. Twenty-three patients with azoospermia and 40 with severe oligozoospermia were analyzed by PCR for the detection of six sequence-tagged sites: sY84 and sY86 for AZFa, sY127 and sY134 for AZFb, and sY254 and sY255 for AZFc. Microdeletions were detected in 28 patients, including 10 azoospermics and 18 severe oligozoospermics. The patients with azoospermia had 43.4% of their microdeletions in the AZFa region, 8.6% in the AZFb region and 17.4% in the AZFc region. In the severe oligozoospermics, 40% were in the AZFa region, 5% in the AZFb region and 5% in the AZFc region. We conclude that microdeletions can be the cause of idiopathic male infertility, supporting conclusions from previous studies.     

Regulation of membrane band 3 Tyr-phosphorylation by proteolysis of p72^syk and possible involvement in senescence process

Erythrocyte senescence is characterized by exposure of cell surface epitopes on cell membrane proteins leading to immune mediated removal of red blood cells. One mechanism for antigen formation is tyrosine phosphorylation （Tyr-P） of the transmembrane protein band 3 by Syk kinase. Our aim was to test the hypothesis that proteolytic activation of Syk kinase by conversion from 72 kDa （p72^syk） to the 36 kDa （p36^syk） isoform enhances its phosphorylating activity independently of the association of Syk kinase with the cytoskeleton. Tyr-P assay was conducted using quantification of 32p uptake into the cytoplasmic domain of band 3 after addition of p72^syk or p36syk. Effect of prephosphorylation of erythrocyte membrane band 3 protein by p36^syk on p72^syk-mediated phosphorylation and the effect of addition of a protease inhibitor （leupeptin） on p72s^yk-mediated phosphorylation were studied by autoradiographic visualization of 32p uptake. Tyr-P by Syk isoforms of membrane skeletal and soluble fractions of band 3 was visualized by immunoblotting. It was found that p36^Syk had a higher band 3 tyrosine phosphorylating activity compared with p72^syk. Pre-phosphorylation with p36^syk or p72^syk increased band 3 phosphorylating activity. Protease inhibition treatment reduced p72^syk but not p36^syk band 3 tyrosine phosphorylating activity significantly. Both soluble and membrane skeletal fractions of band 3 protein were equally tyrosine phosphorylated by each Syk isoform. In conclusion, we confirmed the hypothesis that proteolytic cleavage of p72^syk is an important regulatory step for band 3 Tyr-P and its independence of the association of band 3 with the cytoskeleton.     

DNA damage and biological effects induced by photosensitization with new N(1)-unsubstituted furo[2,3-h]quinolin-2(1H)-ones

New furoquinolinones unsubstituted at the N(1) position were prepared and their photobiological activities were studied in comparison with 4,6,8,9-tetramethylfuro[2,3-h]quinolin-2(1H)-one (HFQ) and 8-MOP. The anti-proliferative activity of furoquinolinones 3a-f was tested upon UVA irradiation in mammalian cells, studying DNA synthesis and clonal growth capacity, and in micro-organisms, evaluating T2 infectivity. Almost all compounds appeared to be more active than 8-MOP, and free of any mutagenic activity and skin phototoxicity. Among them, compound 3b was the most effective one. Similarly to HFQ, compound 3b appeared to be very active also in DNA damaging, forming monoadducts and DPC(L=0), but no ISC and DPC(L>0), both responsible for furocoumarin genotoxicity and phototoxicity. Moreover, Ehrlich ascites cells, photoinactivated by the new furoquinolinone 3b and injected into recipient mice, proved to be capable of inducing protection against a successive challenge performed with the same tumor cells. For all these features, 3b seemed to be a new promising potential drug for PUVA therapy and photopheresis.     

Novel angular furo and thieno-quinolinones: synthesis and preliminary photobiological studies

A number of new furo and thienoquinolinones carrying an electron-withdrawing function or unsubstituted at the position 3 were synthesized in order to obtain new potential photochemotherapeutic agents with increased antiproliferative activity and decreased toxic side effects. Our interest in studying the SAR of these derivatives also prompted us to investigate the influence of N-methylation on biological activity, by preparing N-methyl derivatives. The antiproliferative activity of all the newly synthesized compounds was evaluated and compared to 8-methoxypsoralen (8-MOP), the drug widely used in PUVA-therapy. The 3-unsubstituted thienoquinolinones were generally the most potent derivatives, followed by the furo-analogues. In particular, the unsubstituted thieno[2,3-h]quinoline-2(1H)one showed the highest activity in T2 bacteriophage, HeLa cells and Ehrlich cells tests. All the compounds, assayed on Escherichia coli WP2 TM9, showed a similar mutagenic activity, very close to that of 8-MOP. Except for 2-oxo-1,2-dihydrothieno[2,3-h]quinoline-3-carboxylic acid, which appeared to be very effective, all compounds generated singlet oxygen to slightly larger amounts when compared to 8-MOP. The N-methyl analogues only induced moderate skin erythemas on albino guinea pigs, while all other derivatives appeared to be entirely inactive. On the basis of these results, the unsubstituted thieno[2,3h]quinoline 2(1H)one seems to be the most interesting potential drug for PUVA photochemotherapy and photopheresis.     

Transient lysozymuria during Sarcoma I tumor rejection

The present studies on the in vivo regulation of lysozyme synthesis by cytotoxic macrophages infiltrating Sarcoma I (Sa I) tumor ascites of C57BL/KS mice are an extension of previous observations that the elevation of serum and urinary lysozyme levels might reflect the degree of macrophage-mediated host resistance. Cytotoxic macrophages of host origin are required in the natural rejection of tumors by C57BL/Ks mice challenged with Sa I. The amounts of lysozyme in the serum and urine of the affected mice are quantitated during Sa I growth and rejection. The enzyme levels are correlated with the number of macrophages in the tumor and with the capacity for lysozyme synthesis measured in vitro on monolayers of macrophages isolated from the ascites. The results indicate that the elevation of serum and urinary lysozyme activity is a reflection of both the increased number of cytotoxic macrophages in the host and the qualitative change in the functional state of the cells. These parameters alone cannot account for the extent of the in vivo release of lysozyme in the serum and urine of the affected animals. High levels of lysozyme are present in the extracellular fluids of the mice only when the macrophages are actively participating in host defense. We conclude that in vivo control mechanisms which depend on the presence of viable tumor cells are responsible for the regulation of lysozyme release by cytotoxic macrophages. However, the high levels of lysozyme do not always indicate complete rejection, as indicated by a preliminary observation in which extensive lysozymuria was followed by tumor relapse.     

Band 3 tyr-phosphorylation in normal and glucose-6-phospate dehydrogenase-deficient human erythrocytes

Haemolysis is usually episodic in glucose-6-phosphate dehydrogenase (G6PD) deficiency, often triggered by a period of oxidative stress. In the present work, we investigate a possible biochemical mechanism underlying the enhanced susceptibility of G6PD deficient red blood cells (RBC) to oxidative stress. We analysed eight male subjects with Mediterranean glucose-6P-dehydrogenase deficiency (G6PDd), class II, for their ability in phosphorylating erythrocyte membrane band 3 following oxidative and osmotic stress. Our findings show that this sensitivity is connected to an early membrane band 3 Tyr-phosphorylation in the presence of diamide. However, since both Syk, and Lyn kinases, and SHP-2 phosphatase, mostly implicated in the band 3 P-Tyr level regulation, are alike in content and activity in normal and patient erythrocytes, an alteration in the membrane organization is likely the cause of the anomalous response to the oxidant. We report, in fact, that hypertonic-induced morphological change in G6PDd erythrocyte induces a higher membrane band 3 Tyr-phosphorylation, suggesting a pre-existing membrane alteration, likely due to the chronic lowering of the redox systems in patients. We also report that 1-chloro-2,4-dinitrobenzene-pre-treatment of normal red cells can alter the normal protein-protein and protein-membrane interaction under hypertonic rather than oxidative stress, thus partially resembling the response in patients, and that RBC may utilize a wider range of redox defence, under oxidative conditions, including, but not exclusively, NADPH and glutathione. On the whole, these results would encourage a different approach to the evaluation of the effects of pharmacological administration to patients, giving more attention to the possible drug-induced membrane alteration evidenced by the abnormal band 3 Tyr-phosphorylation.