Effect of temporary meiosis block during prematuration of bovine cumulus–oocyte complexes on pregnancy rates in a commercial setting for in vitro embryo production

Ovum pick up (OPU) associated with in vitro production (IVP) of embryos has been shown as an important tool in cattle breeding to increase the number of descendants from animals of high genetic value. In herds maintained distant from the laboratory, collecting cumulus–oocyte complexes (COCs) and transporting them to the laboratory may take several hours and decrease COCs viability, representing a challenge for commercial settings. In this study, a prematuration culture to induce temporary meiosis block was evaluated in a commercial scale IVP setting as a strategy to transport bovine OPU-derived COCs from Nelore and Brangus donors. Effects on embryo yield and pregnancy rates were assessed. Viable COCs from each donor were destined to one of the experimental groups (control, blocks 1 and 2). Control group COCs were placed in cryotubes with 1 mL TCM199–HEPES. In block groups (1 and 2), COCs were placed in cryotubes with 300 μL TCM 199 + 12 μM butyrolactone I (block medium). All groups were gassed and kept in a thermos bottle for 4 hours at 36 °C. Next, COCs in the control group were transferred to IVM medium and block 1 group to block medium, and cultured for 22 hours and 15 hours, respectively, at 38.5 °C and 5% CO₂ in air. Block 2 COCs were kept in the cryotubes and in the thermos bottle for another 15 hours at 36 °C to simulate long-term transport conditions. After meiosis block in prematuration culture, blocks 1 and 2 COCs were matured in vitro for 22 hours as for the control group. After IVM, COCs in all groups were submitted to IVF and IVC, and blastocyst rates were evaluated on day 7. Embryos were transferred and pregnancy rates evaluated at 60 days of gestation. The mean total number of COCs retrieved by OPU did not differ between Nelore and Brangus donors (16.8 and 17.2, respectively, P > 0.05), but Nelore donors produced more viable COCs than Brangus (10.1 and 7.6, respectively, P < 0.05) and more embryos/cow (3.8 and 2.7, respectively, P < 0.05). Blastocyst rates were similar for control (40.2% and 36.7%), block 1 (37.3% and 34.5%), and block 2 groups (34.7% and 33.6%) for Nelore and Brangus cattle, respectively (P > 0.05). Pregnancy rates did not differ regardless of breed or treatment (36.7%, P > 0.05). In conclusion, temporary meiosis block during prematuration culture did not affect embryo development or pregnancy rates; therefore, this strategy may be used to transport bovine COCs in a commercial IVP setting.     

Preoperative brush and impression cytology in ocular surface squamous neoplasms

OBJECTIVE: Preoperative cytologic diagnoses of ocular surface squamous neoplasms were evaluated and compared with histologic diagnoses. STUDY DESIGN: Impression cytology (Millipore filter paper) and brush cytology were applied to 32 patients who had conjunctival neoplasms. Papanicolaou-stained cytologic preparations and hematoxylin and eosin-stained histologic sections were examined by light microscopy. RESULTS: The brush technique was used on 27 patients; impression cytology was applied in 5 cases. Cytologic and histologic diagnoses were concordant in 26 cases. Squamous cell carcinoma or carcinoma in situ was diagnosed in 18 and dysplasia in 4 cases. Squamous metaplasia and normal-appearing conjunctival epithelial cells were diagnosed cytologically in four cases; of those histologic diagnoses, one was pterygium and three, conjunctival nevus. Four cases revealed discrepancies between the cytologic and histologic preparations. There was one false positive result, and one case was subconjunctival invasion of basal cell carcinoma of the eyelid. CONCLUSION: Impression and brush cytology are fast, cost-effective, reliable and noninvasive diagnostic tools for ocular surface squamous neoplasms. However, the brush technique has several advantages over impression cytology.     

Ferric hydroxamate binding protein FhuD from Escherichia coli: mutants in conserved and non-conserved regions

Uptake of iron complexes into the Gram-negative bacterial cell requires highly specific outer membrane receptors and specific ATP-dependent (ATP-Binding-Cassette (ABC)) transport systems located in the inner membrane. The latter type of import system is characterized by a periplasmic binding protein (BP), integral membrane proteins, and membrane-associated ATP-hydrolyzing proteins. In Gram-positive bacteria lacking the periplasmic space, the binding proteins are lipoproteins tethered to the cytoplasmic membrane. To date, there is little structural information about the components of ABC transport systems involved in iron complex transport. The recently determined structure of the Escherichia coli periplasmic ferric siderophore binding protein FhuD is unique for an ABC transport system (Clarke et al. 2000). Unlike other BP's, FhuD has two domains connected by a long -helix. The ligand binds in a shallow pocket between the two domains. In vivo and in vitro analysis of single amino acid mutants of FhuD identified several residues that are important for proper functioning of the protein. In this study, the mutated residues were mapped to the protein structure to define special areas and specific amino acid residues in E. coli FhuD that are vital for correct protein function. A number of these important residues were localized in conserved regions according to a multiple sequence alignment of E. coli FhuD with other BP's that transport siderophores, heme, and vitamin B₁₂. The alignment and structure prediction of these polypeptides indicate that they form a distinct family of periplasmic binding proteins.     

Analysis of the electroencephalographic activity during the Necker cube reversals by means of the wavelet transform

In previous studies, a perceptual switching related potential was obtained during the observation of a multistable dynamic reversal pattern, where the averaging of the single responses was triggered by subjects pressing a button. The present methodological study aims to increase the signal quality of perceptual switching related potentials considering the dependence of the measurement method on the reaction time of the subject, which may vary significantly during a session, leading to low-amplitude waveform in the averaged event-related-potential (ERP). To overcome this problem in measuring the electrophysiological correlate of an internal event, a pattern selection method based on the wavelet transform (WT) is proposed to choose a subset of single ERPs with more homogenous latencies. Nine subjects observed a Necker cube and were instructed to press the button immediately after perceptual switching. A slow, low-amplitude positive wave with frontocentral amplitude maxima was observed around 250 ms prior to the button press. After the application of a 5 octave WT on single sweeps, the time-frequency coefficients obtained in each octave were averaged across trials. The most dominant feature representing the averaged ERP was the delta (0.5–4 Hz) coefficient occurring between 250 and 125 ms before the button press. By averaging the subset of the single sweeps containing this property, a sharpening and significant amplitude increase of the response peak was observed. Received: 12 September 1997 / Accepted in revised form: 9 July 1998     

Auxin autotrophic tobacco callus tissues resist oxidative stress: the importance of glutathione S-transferase and glutathione peroxidase activities in auxin heterotrophic and autotrophic calli

Auxin autotrophic and heterotrophic tobacco callus lines were grown on MS medium with or without 100 mmol/L NaCl and growth and some of the stress-related activities, such as GPX, SOD, CAT, GST, GSH-PX, as well as the concentration of ethylene and H2O2, were measured and compared with each other. The auxin autotrophic calli grew slower, however, on the NaCl-containing medium the growth rate was higher than that of the heterotrophic cultures after two weeks of culturing. The stress-related ethylene production was lower in the autotrophic cultures and, contrary to the heterotrophic tissues, its level did not change significantly upon NaCl treatment. The guaiacol peroxidase (GPX) activities were higher in the autotrophic tissues in all cell fractions regardless of the presence of NaCl. Treated with NaCl, the GPX activities elevated in the soluble and covalently-bound fractions in the heterotrophic calli, but were not further increased in the autotrophic line. SOD and CAT activities were higher in the heterotrophic tissues, and were increased further by 100 mmol/L NaCl treatment. The GST and GSH-PX activities were higher in the autotrophic line, which might explain their enhanced stress tolerance. In the autotrophic tissues, the elevated antioxidant activities led to reduced levels of H2O2 and malondialdehyde; under mild NaCl stress, these levels decreased further. The lower growth rate and the effective protection against NaCl stress-induced oxidative damage of the autotrophic line can be explained by the cell wall-bound peroxidase and GSH-PX activities in the auxin autotrophic tissues. Their maintained growth rate indicates that the autotropic cultures were more resistant to exogenous H2O2.     

Fine needle aspiration cytology of ovarian lesions

OBJECTIVE: To assess the diagnostic value of fine needle aspiration cytology (FNAC) in ovarian lesions. STUDY DESIGN: Sixty-two fine needle aspirations of varied ovarian masses were performed between January 2000 and February 2003. Aspiration material was obtained from fresh tissues at the time of frozen section before dissection of the specimens. The slides were evaluated by a pathologist who was blinded to the gross findings and histopathologic diagnosis. RESULTS: A total of 62.9% of cases were assessed as malignant and 30.6% as benign. Two false positive and 2 false negative cases occurred. In our series the overall sensitivity and specificity were 95.1% and 90.4%, respectively. Seven benign and 15 malignant lesions could be subclassified specifically. CONCLUSION: We studied excised specimens, but since ovarian masses are reachable by laparoscopically and ultrasonographically guided aspiration, FNAC can be used for diagnoses of these lesions. If a multidisciplinary approach can be carried out in patients with ovarian lesions, cytopathologic interpretation can provide optimum benefits.