The oxidizing agent tertiary butyl hydroperoxide induces disturbances in spindle organization, c-meiosis, and aneuploidy in mouse oocytes

It has been recently proposed that a concomitant generationof oxidative stress of oocytes with increasing maternal agemay be a major factor responsible for the age-related increasein aneuploid conceptions. As a preliminary step in the testingof this hypothesis, we need to confirm that oxidative stressin itself can induce errors in chromosome segregation. In orderto achieve this goal, germinal vesicle (GV)-stage mouse oocytesfrom unstimulated ICR and (C57BLxCBA) F₁ hybrid female micewere matured in vitro for 9 h for metaphase I (MI) oocytes or16 h for metaphase II (MII) oocytes in the presence of varyingconcentrations of the oxidizing agent tertiary-butyl hydroperoxide(tBH). MII oocytes from (C57BLxCBA) F₁ hybrid mice were fixedand C-banded for karyotyping analysis. MI and MII oocytes fromICR mice were fixed and stained with the DNAfluorescent probe4',6-diamidino-2-phenylindole (DAPI) to detect abnormalitiesin chromosomal distribution. Meiosis I and meiosis II spindlesfrom ICR mice were visualized by confocal immunofluorescencemicroscopy. Data from these experiments demonstrate that in-vitroexposure of mouse oocytes to tBH during meiosis I reduces thelength (pole-to-pole distance) and width (diameter at the equatorof the spindle) of meiosis I and meiosis II spindles. This reductionis associated with an increase in the percentage of oocytesshowing chromosome scattering and clumping on the MII plate,and of aneuploidy (hyperhaploidy) in MII oocytes. However, tBHat the concentrations used in the present study has only a minimalnegative effect on the frequency of meiotic maturation. Theseresults suggest that oxidative stress during meiotic maturationin vitro may induce chromosomal errors that are undetectablein the living oocyte and whose developmental consequences maybecome manifest after fertilization. aneuploidy/meiosis/mouse oxidative stress/spindle/tertiary butyl hydroperoxide     

Immunogenicity and T cell recognition in swine of foot-and-mouth disease virus polymerase 3D

Immunization of domestic pigs with a vaccinia virus (VV) recombinant expressing foot-and-mouth disease virus (FMDV) 3D protein conferred partial protection against challenge with infectious virus. The severity reduction of the clinical symptoms developed by the challenged animals occurred in the absence of significant levels of anti-3D circulating antibodies. This observation suggested that the partial protection observed was mediated by the induction of a 3D-specific cellular immune response. To gain information on the T cell recognition of FMDV 3D protein, we conducted in vitro proliferative assays using lymphocytes from outbred pigs experimentally infected with FMDV and 90 overlapping peptides spanning the complete 3D sequence. The use of pools of two to three peptides allowed the identification of T cell epitopes that were efficiently recognized by lymphocytes from at least four of the five animals analyzed. This recognition was heterotypic because anti-peptide responses increased upon reinfection of animals with a FMDV isolate from a different serotype. The results obtained with individual peptides confirmed the antigenicity observed with peptide pools. Detection of cytokine mRNAs by RT-PCR in lymphocytes stimulated in vitro by individual 3D peptides revealed that IFN-gamma mRNA was the most consistently induced, suggesting that the activated T cells belong to the Th 1 subset. These results indicate that 3D protein contains epitopes that can be efficiently recognized by porcine T lymphocytes from different infected animals, both upon primary and secondary (heterotypic) FMDV infection. These epitopes can extend the repertoire of viral T cell epitopes to be included in subunit and synthetic FMD vaccines.     

Factor Structure, Reliability, and Validity of the Pain Catastrophizing Scale

The Pain Catastrophizing Scale (PCS; Sullivan et al., Psychol. Assess. 7, 524–532, 1995) has recently been developed to assess three components of catastrophizing: rumination, magnification, and helplessness. We conducted three studies to evaluate the factor structure, reliability, and validity of the PCS. In Study I, we conducted principal-components analysis with oblique rotation to replicate the three factors of the PCS. Gender differences on the original PCS subscales were also analyzed. In Study II, we conducted confirmatory factor analyses to evaluate the adequacy of fit of four alternative models. We also evaluated evidence for concurrent and discriminant validity. In Study III, we evaluated the ability of the PCS and subscales to differentiate between the responses of clinic (students seeking treatment) and nonclinic undergraduate samples. Also, in the clinic sample, we evaluated evidence of concurrent and predictive validity for the PCS. The internal consistency reliability indices for the total PCS and subscales were examined in all three studies. Limitations and future directions are discussed.     

Inhibition of nitric oxide synthase increases synaptophysin mRNA expression in the hippocampal formation of rats

Synaptophysin is a protein involved in the biogenesis of synaptic vesicles and budding. It has been used as an important tool to investigate plastic effects on synaptic transmission. Nitric oxide (NO) can influence plastic changes in specific brain regions related to cognition and emotion. Experimental evidence suggests that NO and synaptophysin are co-localized in several brain regions and that NO may change synaptophysin expression. Therefore, the aim of the present work was to investigate if inhibition of NO formation would change synaptophysin mRNA expression in the hippocampal formation. Male Wistar rats received single or repeated (once a day for 4 days) i.p. injections of saline or l-nitro-arginine (l-NOARG, 40 mg/kg), a non-selective inhibitor of nitric oxide synthase (NOS). Twenty-four hours after the last injection the animals were sacrificed and their brains removed for ‘in situ’ hybridization study using ³⁵S-labeled oligonucleotide probe complementary to synaptophysin mRNA. The results were analyzed by computerized densitometry. Acute administration of l-NOARG induced a significant (p < 0.05, ANOVA) increase in synaptophysin mRNA expression in the dentate gyrus, CA1 and CA3. The effect disappeared after repeated drug administration. No change was found in the striatum, cingulated cortex, substantia nigra or nucleus accumbens. These results reinforce the proposal that nitric oxide is involved in plastic events in the hippocampus.     

Experimental autoimmune myasthenia gravis in naive non-obese diabetic (NOD/LtJ) mice: susceptibility associated with natural IgG antibodies to the acetylcholine receptor

Naive non-obese diabetic (NOD/LtJ) mice spontaneously produce natural IgG autoantibodies against self-antigens associated with the experimental autoimmune diseases to which they are susceptible: insulin-dependant diabetes mellitus, systemic lupus erythematosus and experimental autoimmune encephalomyelitis. We discovered recently that NOD/LtJ mice also spontaneously produce IgG antibodies to the acetylcholine receptor (AchR), an antigen that can induce experimental autoimmune myasthenia gravis (EAMG) in susceptible rodents. However, there are no reports indicating that NOD/LtJ mice are susceptible to EAMG. To test whether the presence of spontaneous IgG autoantibodies can predict susceptibility to an autoimmune disease, we challenged NOD/LtJ mice using a standard protocol to induce EAMG. We now report that NOD/LtJ mice developed EAMG, although to a somewhat lesser degree than did C57BL/6 mice, a strain regarded as highly susceptible to the disease. Both strains produced comparable levels of immune antibodies to AchR of the complement-fixing isotypes IgG2a and IgG2b; however, NOD/LtJ mice produced significantly more IgG1. An antigen-specific T cell proliferative response to AchR of the same magnitude was detected in both strains, together with the secretion of similar amounts of IFN-gamma. Thus, NOD/LtJ mice are susceptible to EAMG and disease induction is accompanied by immune responses comparable to those seen in the susceptible strain C57BL/6. These results support the association between specific, natural IgG autoantibodies and susceptibility to the induction of a particular autoimmune disease.     

Induction of interleukin 2 (IL2) and interferon-γ and enhancement of IL2 receptor expression by a CD26 monoclonal antibody

The ability of the 134-2C2 monoclonal antibody (mAb; CD26) to transmit an activation signal and to affect T cell proliferation has been studied. The 134-2C2 mAb, although not being mitogenic by itself, is able to increase the proliferation of purified T cells in the presence of exogenous interleukin 2 (IL2) or phorbol 12-myristate 13-acetate (PMA). No effect of our mAb was observed on the proliferation of T cells induced by other stimuli such as Sepharose-bound CD3 mAb, phytohemagglutinin or calcium ionophore. Since the co-stimulatory effect of 134-2C2 mAb on PMA-induced T cell proliferation was strongly inhibited by an anti-Tac antibody, its involvement on the IL2/IL2 receptor pathway was investigated. An increased IL2 secretion in T cells cultured with PMA plus 134-2C2 mAb was observed and Northern blot analysis showed that the mAb 134-2C2 acts synergistically with PMA favoring the induction of both IL2 and interferon-γ mRNA synthesis, as well as the enhancement of IL2 receptor and transferrin receptor mRNA expression. Studies on mechanisms implicated in signal transduction showed that 134-2C2 mAb modifies neither intracellular calcium levels nor phosphoinositide breakdown. Additionally, no effect was exerted on protein kinase C translocation. These data suggest that the CD26 antigen is involved in T cell activation in an IL2/IL2 receptor-dependent pathway.     

Analysis of human papillomavirus prevalence and TP53 polymorphism in head and neck squamous cell carcinomas

Head and neck squamous cell carcinoma is a disease associated with tobacco and alcohol abuse. There is evidence that the oncogenic human papillomavirus (HPV) may also be a risk for upper aerodigestive tract cancers. High-risk HPVs encode two early proteins, E6 and E7, that can bind to p53 and pRb, respectively, and induce its degradation or inactivation. The TP53 gene has a single polymorphism at codon 72 of exon 4 that encodes either arginine (Arg) or proline (Pro). The purpose of this study was to evaluate the role of HPV infection and TP53 polymorphism in head and neck cancer. We analyzed 50 tumors, as well swabs of oral mucosa from 142 control individuals, with a polymerase chain reaction technique. The prevalence of HPV in controls was 10.6% and in cancer specimens 16%. The frequency distribution of genotypes in controls was 50% Arg/Arg, 43% Arg/Pro and 7% Pro/Pro; in tumors, it was 52% Arg/Arg, 32% Arg/Pro, and 16% Pro/Pro. Contrary to the results of some studies on cervical cancer, no association between any TP53 genotype or allele and the development of head and neck cancer was observed, regardless of HPV status, except for the Pro/Pro genotype, which is associated with the absence of HPV. The arginine allele appears to protect against head and neck cancers. Also, the data showed that HPV infection results in no increased risk of developing head and neck tumors.     

Yolk sac tumors with pure and mixed polyvesicular vitelline patterns

Four cases of polyvesicular vitelline tumor are presented; two were of a previously unreported pure type, and the other two were mixed with endodermal sinus tumor.The morphologic features of the vesicles favor an endodermal origin, as originally proposed by Teilum. Marked specialization of the vesicular lining cells, seen ultrastructurally, suggests a differentiation toward gut structures and mature yolk sac. One case of pure polyvesicular vitelline tumor showed massive erythropoiesis. We propose that the pure tumor reflects an intermediate degree of differentiation within the selectively endodermal yolk sac tumor group, that is, a further stage of organization than the endodermal sinus tumor.In our cases of pure polyvesicular vitelline tumor, the marked degree of differentiation was correlated with an improved prognosis, as in the case of the possible homologue of this tumor, the yolk sac tumor of the infant testis. In contrast, the two cases of the tumor admixed with endodermal sinus tumor illustrated the low survival rate expected in the pure endodermal sinus tumor; in these cases the metastases had no polyvesicular component. Because of the significance of such a difference in prognosis we emphasize the importance of an accurate diagnosis, suggesting that a large number of sections be taken in order to demonstrate any endodermal sinus tumor component that may be present, and that the possibility of pure polyvesicular vitelline tumor always be considered in the differential diagnosis of multicystic ovarian tumors.     

ICAM-3, the third LFA-1 counterreceptor,is a co-stimulatory molecule for both resting and activated T lymphocytes

Optimal activation of human T cells mediated by ligation of CD3/T cell receptor (TcR) complex requires co-stimulatory signals. These can be provided by the adhesive interaction between receptor molecules on T cells and their counter-receptors on antigen-presenting cells. Soluble ICAM-3, anti-ICAM-3 and anti-CD3 mAb were utilized to address the role of the ICAM-3/LFA-1 pathway in TcR/CD3-dependent or -independent T cell activation. Immunoaffinity-purified ICAM-3 co-immobilized with suboptimal concentrations of anti-CD3 monoclonal antibody (mAb) stimulated T lymphocytes as monitored by the expression of the lymphocyte activation antigens CD25 and CD69. The mechanism underlaying this activation appear to involve the interaction of ICAM-3 with a β2 integrin, likely to be LFA-1, since mAb to the CD18 chain completely inhibited T cell activation. Similar experiments demonstrated that anti-ICAM-3 mAb were able to co-stimulate both resting (cord blood) and activated (T cell clones) T lymphocytes. On the contrary, anti-ICAM-1 mAb were only co-stimulatory for CD25 expression on activated but not on resting T cells. In addition, we have found that some γδ T cell clones bearing the Vδ1 segment were activated by direct mAb engagement of ICAM-3 in the absence of TcR/CD3 occupancy. Furthermore, immobilized anti-ICAM-3 mAb also induced development of dentritic processes. In conclusion, our data suggest that ICAM-3 on the surface of both T cells and antigen-presenting cells plays an essential role in the initiation of the immune response.     

Endophytic bacteria in Coffea arabica L

Eighty-seven culturable endophytic bacterial isolates in 19 genera were obtained from coffee plants collected in Colombia (n = 67), Hawaii (n = 17), and Mexico (n = 3). Both Gram positive and Gram negative bacteria were isolated, with a greater percentage (68%) being Gram negative. Tissues yielding bacterial endophytes included adult plant leaves, various parts of the berry (e.g., crown, pulp, peduncle and seed), and leaves, stems, and roots of seedlings. Some of the bacteria also occurred as epiphytes. The highest number of bacteria among the berry tissues sampled was isolated from the seed, and includes Bacillus , Burkholderia , Clavibacter , Curtobacterium , Escherichia , Micrococcus , Pantoea , Pseudomonas , Serratia , and Stenotrophomonas . This is the first survey of the endophytic bacteria diversity in various coffee tissues, and the first study reporting endophytic bacteria in coffee seeds. The possible role for these bacteria in the biology of the coffee plant remains unknown.