Bovine oocytes and early embryos express Staufen and ELAVL RNA-binding proteins.

RNA-binding proteins (RBP) influence RNA editing, localization, stability and translation and may contribute to oocyte developmental competence by regulating the stability and turnover of oogenetic mRNAs. The expression of Staufen 1 and 2 and ELAVL1, ELAVL2 RNA-binding proteins during cow early development was characterized. Cumulus-oocyte complexes were collected from slaughterhouse ovaries, matured, inseminated and subjected to embryo culture in vitro. Oocyte or preimplantation embryo pools were processed for RT-PCR and whole-mount immunofluorescence analysis of mRNA expression and protein distribution. STAU1 and STAU2 and ELAVL1 mRNAs and proteins were detected throughout cow preimplantation development from the germinal vesicle (GV) oocyte to the blastocyst stage. ELAVL2 mRNAs were detectable from the GV to the morula stage, whereas ELAVL2 protein was in all stages examined and localized to both cytoplasm and nuclei. The findings provide a foundation for investigating the role of RBPs during mammalian oocyte maturation and early embryogenesis.

Tenoxicam 20 mg or 40 mg after thoracotomy: a prospective, randomized, double-blind, placebo-controlled study.

Forty-five adults undergoing thoracotomy were randomized to receive placebo, tenoxicam 20 mg or tenoxicam 40 mg IV during chest wall closure. All patients received intraoperative fentanyl and intercostal blocks followed by morphine by patient-controlled analgesia. Patient numbers 13 to 45 also received thoracic epidural analgesia by continuous infusion of bupivacaine 0.125%, patient numbers 25 to 45 having fentanyl 2 microg/ml added to the epidural infusion. Efficacy parameters and adverse reactions were assessed over the first 24 hours postoperatively. On a 100 mm visual analogue scale, mean (SD) pain at rest (adjusted area under curve for hours 1 to 24) was 25.8 (12.5), 17.4 (14.8) and 16.5 (13.3) mm for groups receiving placebo, 20 mg and 40 mg tenoxicam, respectively (ANOVA: P<0.05). There were no significant differences between study groups postoperatively in pain on coughing, opioid consumption, blood gas measurements, nausea, vomiting, sedation, blood loss, haemoglobin or serum creatinine. One patient in each tenoxicam group reported epigastric pain, rated moderate. These data support the inclusion of tenoxicam 20 mg IV in the management of pain at rest for patients undergoing thoracotomy, but do not show additional benefit for a higher dose.

Expression of the bovine oestrogen receptor-beta (bERbeta) messenger ribonucleic acid (mRNA) during the first ovarian follicular wave and lack of change in the expression of bERbeta mRNA of second wave follicles after LH infusion into cows.

In a previous study, the ERbeta cDNA protein-coding region was utilised to clone bovine ERbeta. The objectives in this study were to examine (1) ERbeta mRNA expression in ovarian follicles throughout the bovine first follicular wave, and (2) effect of LH infusion into cows on bERbeta mRNA expression during the second follicular wave. In experiment 1, heifers (4-5 per time point) were ovariectomized at 12, 24, 36, 48, 60, 72, 84, 96, 144, or 216 h after emergence of the first follicular wave after oestrus. In experiment 2, saline or LH was pulsed hourly (computer-controlled syringe pump) into cows (n = 31; 5-6 per treatment) at wave emergence for 2 or 4 days: wave 1-saline (W1S), wave 2-saline (W2S), or wave 2-LH (25 microg/h; W2LH). Ovaries were removed on day 2 or day 4 after wave emergence. Follicles, 2-19mm in size, were dissected, frozen, and stored at -80 degrees C for in situ hybridisation with two bERbeta cRNA probes. Expression of bERbeta mRNA was localised in granulosa cells of healthy follicles. In experiment 1, bERbeta mRNA expression did not change with time points of the wave showing no association of bERbeta mRNA expression with follicular selection and dominance. However, bERbeta mRNA expression decreased with increase in size of all follicles. Expression of bERbeta mRNA was greater in very small follicles (2-4 mm) than in large (> or = 9 mm) follicles. In experiment 2, expression of bERbeta mRNA in follicles did not differ either between W1S and W2S or between W2S and W2LH. In summary, bERbeta mRNA expression decreased with increasing follicular size. However, neither stage of the wave (selection or dominance), nor pulsatile infusion of LH influenced bERbeta mRNA expression.

Concentrations of steroids and expression of messenger RNA for steroidogenic enzymes and gonadotropin receptors in bovine ovarian follicles of first and second waves and changes in second wave follicles after pulsatile LH infusion.

The objectives were to compare expression of mRNA for cytochrome P450 cholesterol side-chain cleavage (P450scc), cytochrome P450 17alpha-hydroxylase (P450c17), cytochrome P450 aromatase (P450arom), 3beta-hydroxysteroid dehydrogenase Delta(4), Delta(5) isomerase (3beta-HSD), FSH receptor (FSHr) and LH receptor (LHr) in bovine ovarian follicles of the first and second waves of the bovine oestrous cycle and to determine if LH infusion changes growth, steroidogenesis and gene expression in second wave follicles. Transrectal ultrasonography was used to examine follicular size changes during the oestrous cycle in non-lactating Holstein cows (n=31). Saline or purified bovine LH was infused intravenously into cows at emergence of follicular waves for 2 or 4 days using a computer-controlled syringe pump (n=5-6 per treatment). Treatments were: wave 1, saline (W1S); wave 2, saline (W2S) or LH (25 microg/h; W2LH). During infusion, blood samples were collected at 12min intervals for 8h via i.v. catheters for measurement of serum LH concentrations. Ovaries were removed from cows on days 2 or 4 after emergence of follicular waves. Follicles were frozen and stored at -80 degrees C. Follicular fluid (FF, 50 microl) was collected for determination of progesterone (P4), oestradiol-17beta (E2) and androstenedione (A4) concentrations. Frozen sections (14 microm) were used for in situ hybridization to measure expression of mRNA (% pixel intensity) for P450scc, P450c17, P450arom, 3beta-HSD, FSHr, and LHr. LH infusion resulted in a serum LH pattern (high frequency) similar to the early luteal phase. There were no significant differences in size of follicles among the three treatment groups. Follicular fluid concentrations of E2 and A4 in W2S were lower than those of W1S on day 2 of a follicular wave. LH infusion into cows during the midluteal phase increased follicular fluid E2 and A4 concentrations in second wave follicles on day 2 of a follicular wave (W2LH) compared to those of W2S. The increase in follicular fluid E2 on day 2 in wave 2 follicles after LH infusion occurred possibly through an increase in mRNA expression of P450c17 and 3beta-HSD. In conclusion, follicular fluid concentrations of E2 and A4 were lower in W2S than in W1S and E2 and A4 concentrations were restored by infusion of LH in W2LH with an increase in mRNA expression of P450c17 and 3beta-HSD.

Dominant bovine ovarian follicular cysts express increased levels of messenger RNAs for luteinizing hormone receptor and 3 beta-hydroxysteroid dehydrogenase delta(4),delta(5) isomerase compared to normal dominant follicles.

The objective was to compare ovarian steroids and expression of mRNAs encoding cytochrome P450 side-chain cleavage, cytochrome P450 17 alpha-hydroxylase, cytochrome P450 aromatase, 3 beta-hydroxysteroid dehydrogenase Delta(4),Delta(5) isomerase, LH, and FSH receptors and estrogen receptor-beta in ovaries of cows with dominant and nondominant ovarian follicular cysts and in normal dominant follicles. Estradiol-17 beta, progesterone, and androstenedione concentrations were determined in follicular fluid using specific RIAs. Dominant cysts were larger than young cysts or dominant follicles, whereas nondominant cysts were intermediate. Estradiol-17 beta (ng/ml) and total steroids (ng/follicle) were higher in dominant cysts than in dominant follicles. Expression of LH receptor and 3 beta-hydroxysteroid dehydrogenase mRNAs was higher in granulosa cells of dominant cysts than in dominant follicles. Nondominant cysts had higher follicular concentrations of progesterone, lower estradiol-17 beta concentrations, and lower expression of steroidogenic enzyme, gonadotropin receptor, and estrogen receptor-beta mRNAs than other groups. In summary, increased expression of LH receptor and 3 beta-hydroxysteroid dehydrogenase mRNAs in granulosa and increased follicular estradiol-17 beta concentrations were associated with dominant cysts compared to dominant follicles. Study of cysts at known developmental stages is useful in identifying alterations in follicular steroidogenesis.

Cyclooxygenase-2 and prostaglandin E(2)(PGE(2)) receptor messenger RNAs are affected by bovine oocyte maturation time and cumulus-oocyte complex quality, and PGE(2) induces moderate expansion of the bovine cumulus in vitro.

Expression of cyclooxygenase-2 (COX-2) and prostaglandin E(2) (PGE(2)) receptor 2 (EP2) are necessary for rodent cumulus expansion in vivo. Prostaglandin E(2) receptor 3 (EP3) has been detected in bovine preovulatory follicles and corpora lutea. The current experiments examined the effect of PGE(2) on bovine cumulus expansion in vitro and expression of COX-2, EP1, EP2, EP3, and EP4 mRNAs in bovine cumulus-oocyte complexes (COCs) at 0, 6, 12, 18, and 24 h time points during maturation in vitro. Concentrations of PGE(2) above 50 ng/ml resulted in moderate cumulus expansion of bovine COCs, but expansion did not occur in the absence of serum. COX-2 mRNA expression increased in bovine COCs at 6 h and 12 h of maturation, then decreased. EP2 mRNA was detectable by reverse transcription-polymerase chain reaction at all time points. EP3 mRNA expression increased in COCs from 0 to 6 h and remained at this higher level through the culture period. Very low levels of EP4 mRNA expression were detectable, but EP1 was not detected in bovine COCs. Because EP receptor mRNAs and COX-2 mRNA are expressed in bovine COCs, there exists the potential for a prostaglandin autocrine/paracrine regulatory pathway during oocyte maturation. Differential expression of the EP3 mRNA among varying COC classes indicates that this gene product may be a useful marker of oocyte competence. Although the PGE(2) pathway is involved in cumulus expansion, serum factors are required to mediate PGE(2)-induced expansion.

Administration of progesterone to cows with ovarian follicular cysts results in a reduction in mean LH and LH pulse frequency and initiates ovulatory follicular growth.

Cows with ovarian follicular cysts were treated with progesterone to determine whether a reduction in LH concentrations and initiation of ovulatory follicular waves would occur. Cysts were diagnosed using transrectal ultrasonography when single follicular structures > 20 mm or multiple structures > 15 mm in diameter were present for 7 d in the presence of low progesterone concentrations. Three groups were studied: 1) cows with normal estrous cycles (CYC, n = 8); 2) cows with untreated cysts (CYST, n = 7); and 3) cows with cysts treated with two progesterone-releasing intravaginal devices (PRID, n = 8) for 9 d. Ovaries were examined with transrectal ultrasonography, and blood samples were collected daily for analysis of progesterone and FSH. Serial blood samples for determination of mean LH and LH pulse frequency were collected on d 0 (CYST and PRID cows only), 1, 5, 9, and 10. Progesterone concentrations were higher in PRID cows than in CYST cows throughout the PRID treatment period (P < .002). On d 0, LH pulse frequency was similar (P = .10) in PRID (6.6+/-.6 pulses/8 h) and CYST cows (5.1+/-.6 pulses/8 h), but mean LH tended to be higher (P = .054) on d 0 in PRID cows (2.5+/-.2 ng/mL) than in CYST cows (1.9+/-.2 ng/mL). Mean LH and LH pulse frequency decreased (P < .002) by d 1 in PRID cows (1.1+/-.2 ng/mL, 1.8+/-.6 pulses/8 h) compared with CYST cows (2.1+/-.2 ng/mL, 5.6+/-.6 pulses/8 h) and remained lower throughout most of the experimental period. The FSH concentrations were higher (P < .01) in PRID cows than in CYC and CYST cows on d 3 and 4. The increase in FSH concentrations preceded emergence of the PRID-induced follicular wave. All PRID cows and four of seven CYST cows initiated new follicular waves during the period of PRID treatment. Follicular waves were initiated later (P < .05) in CYST cows (d 5.2+/-1.7) and PRID cows (d 5.5+/-.6) than in CYC cows (d 1.8+/-.3). Cysts were smaller (P < .01) at the end of the treatment period in PRID cows compared with CYST cows. No CYST cows ovulated, but all PRID cows ovulated newly developed follicles 3 or 4 d after PRID removal. Treatment with exogenous progesterone reduced LH in cows with cysts, and this was followed by development of normal ovulatory follicles.

Cloning, sequencing, and localization of bovine estrogen receptor-beta within the ovarian follicle.

The potential role of estrogen receptor-beta (ERbeta) in normal ovarian folliculogenesis and in reproductive disorders such as ovarian follicular cysts has not been well defined. Therefore, we were interested in cloning, sequencing, and localizing ERbeta mRNA and protein within the bovine ovary. Bovine ERbeta (bERbeta) was amplified by reverse transcription-polymerase chain reaction (RT-PCR), then cloned and sequenced. Results showed that the open reading frame of bERbeta cDNA spanned 1584 nucleotides encoding a protein of 527 amino acids. The N-terminal region of bERbeta was found to be 80% homologous to human and mouse ERbeta and 79% homologous to rat ERbeta. Bovine ERbeta DNA-binding domain was 100% homologous to human, mouse, and rat ERbeta sequences. The C-terminal/ligand-binding domain of bERbeta was 89% homologous to human, 86% homologous to mouse, and 88% homologous to rat ERbeta. Human and bovine ERbeta amino acid sequences are similar in that their coding region extended farther 5' than initially reported for the published rat ERbeta sequence. Using in situ hybridization and immunohistochemistry, ERbeta mRNA and protein, respectively, were demonstrated to be present in granulosa cells of antral follicles in various stages of follicular growth. These findings suggest a role for bERbeta in ovarian follicular growth and maturation.

Expression of steroidogenic acute regulatory protein messenger ribonucleic acid is limited to theca of healthy bovine follicles collected during recruitment, selection, and dominance of follicles of the first follicular wave.

Expression of mRNA encoding steroidogenic acute regulatory protein (StAR) in bovine follicles during recruitment and selection was examined. Dairy heifers (4-5/time period) were ovariectomized at 12, 24, 36, 48, 60, 72, 84, or 96 h after initiation of the first follicular wave (Time 0) after estrus. Follicles were collected and stored at -80 degrees C until sectioning. Expression of StAR mRNA was localized by in situ hybridization and quantified by image analysis. Expression of StAR mRNA was first detected in theca interna of antral follicles as small as 0.5 mm in diameter and increased with increasing follicular size (>/= 4 mm; r = 0.75; p < 0.001). StAR mRNA was undetectable in granulosa of healthy follicles at any size or stage of follicular wave examined. However, granulosa or luteinized granulosa of some advanced or late atretic follicles expressed StAR mRNA. During recruitment, StAR mRNA expression in theca cells was similar among recruited follicles (4-8 mm). During selection of dominant follicles (36-48 h), StAR mRNA was expressed in theca of more than one follicle (7-9 mm); therefore, expression of StAR mRNA may not be associated with dominant follicle selection. StAR mRNA in theca was higher (p < 0.05) at 48 h after initiation of the first follicular wave than at 12, 24, and 36 h, and it remained elevated thereafter through 96 h. Dominant follicles expressed more (p < 0.01) StAR mRNA in theca than did subordinate healthy follicles. Healthy follicles expressed higher (p < 0.05) StAR mRNA in theca than atretic follicles. In summary, levels of StAR mRNA increased in theca with stage of follicular wave and size of follicles. Follicular atresia was associated with reduced expression of StAR mRNA in theca cells. The results indicate that expression of StAR mRNA in theca may not be the primary limiting factor for follicular recruitment and selection.

Tenoxicam and paracetamol-codeine combination after oral surgery: a prospective, randomized, double-blind, placebo-controlled study.

We studied 90 adults undergoing surgical removal of at least both lower third molar teeth as day cases under standardized general anaesthesia. Patients were allocated randomly (with stratification for surgeon) to receive tenoxicam 40 mg, tenoxicam 20 mg or placebo i.v. at induction of anaesthesia and orally (effervescent tablets) with food on each of the subsequent 2 days. Panadeine (paracetamol 500 mg-codeine 8 mg) was given before operation and was available as needed for pain thereafter, to a limit of two tablets every 4 h. Nefopam i.v. was also available. Efficacy variables and adverse reactions were assessed over 6 days. Over the 6-day period, patients who received tenoxicam reported less pain on rest (area under the curve; P < 0.05) and less disturbance in sleep (P < 0.01) even though they used fewer Panadeine tablets (P < 0.05). Differences between tenoxicam 40 mg and 20 mg were not significant. There was no significant difference in nefopam requirements or side effects, and no adverse event attributable to the study medication.

Superovulatory responses in dairy cows following ovulation of the dominant follicle of the first wave.

In the present study we investigated the effect of hCG administration on Day 7 (Day 0 = day of standing estrus) to ovulate the dominant follicle of the first wave and the associated increase in progesterone concentration on subsequent superovulatory response in dairy cows. Twenty cyclic lactating cows were allocated at random to 2 groups: control (n = 10) and hCG-treated (n = 10). The ovaries of each cow were scanned using an ultrasound scanner on Day 7, to confirm the presence of the dominant follicle and thereafter every other day until embryo recovery. All cows received a total dose of 400 mg Folltropin-V in decreasing amounts for 5 days (Days 9 to 13) and 35 mg PGF(2alpha) on Day 12. In addition, the treated cows received 1000 IU hCG on Day 7. All cows were inseminated twice during estrus, and the embryos were collected 7 days later by a nonsurgical procedure. Blood smaples were taken at different times of the treatment period for progesterone determination. All cows possessed a dominant follicle at Day 7, and all but one of the hCG-treated cows ovulated the dominant follicle and formed an accessory corpus luteum. Plasma progesterone concentrations were significantly higher (P<0.01) in hCG-treated cows than control cows on the first day of Folltropin treatment and on the day of PGF(2alpha) injection. The mean number of follicles at estrus, the number of ovulations, the total number of embryos and the number of transferable embryos were not different (P>0.05) between control and hCG-treated cows.

Follicular growth, ovulation and embryo recovery in dairy cows given FSH at the beginning or middle of the estrous cycle.

Variability in the superovulation response is an important problem for the embryo transfer industry. The objective of this study was to determine whether FSH treatment at the beginning of the cycle would improve the ovulation rate and embryo yield in dairy cows. Twenty-eight postpartum cyclic dairy cows were allocated at random to 4 treatment groups (A, B, C and D). Group A cows (n=10) received FSH (35 mg) at a decreasing dose, starting on Day 9 (Day 0=day of estrus) for 5 days followed by PGF2alpha (35 mg) on Day 12. Cows assigned to Groups B, C and D (n=6 cows each, respectively) were given 35 mg FSH at a decreasing dose from Days 2 to 6 followed by PGF2alpha on Day 7. Group C and D cows received PRID inserts from Day 3 to Day 7. Cows in Group D additionally received 1000 IU hCG 60 hours after PGF2alpha treatment. Ovaries were scanned daily using a real time ultrasound scanner from the beginning of FSH treatment until embryo recovery, to monitor follicular development, ovulation and the number of unovulated follicles. Embryos were recovered from the uterus by a nonsurgical flushing technique 7 days after breeding. There were no differences (P>0.01) in the number of follicles>10 mm at 48 hours after PGF2alpha treatment among the 4 groups. The mean numbers of follicles were 10.6+/-1.2, 9.3+/-1.3, 12.2+/-1.3 and 15.0+/-2.9 for Groups A, B, C and D, respectively. A significantly (P<0.001) higher number of ovulations was observed and a larger number of embryos was recovered in Group A than in the other groups. The results of this study indicate that superovulation with FSH at the beginning of the cycle causes sufficient follicular development but results in very low ovulation and embryo recovery rates.

ESCA investigation of low-temperature ammonia plasma-treated polyethylene substrate for immobilization of protein.

A low-temperature radiofrequency plasma excited in anhydrous ammonia was used to modify polyethylene substrate surfaces for covalent immobilization of proteins. Electron spectroscopy for chemical application (ESCA) was used for surface characterization of polyethylene to a depth scale of 7 nm. The data revealed that surface modification is extensive and occurs in seconds at low discharge power. Primary amino functionalities were detected on the polyethylene surface and the level is dependent on plasma parameters. 125I-labelled antibodies covalently attached to amino groups via glutaraldehyde allowed the conditions for optimum level of primary amine to be established. Both ESCA data and protein loadings are in excellent agreement.

Apparatus for the electrical characterisation of conductive fluids.

Non-invasive and fully automated conductimetric measurements of electrolyte and bacterial samples were achieved in a closed volume test cell, comprising a magnetic field coil and detector. By monitoring field induced currents in sample electrolytes the magnitude of the sample current was shown to vary as the inverse of the sample impedance. The impedance characteristic was shown to be that of an LCR resonant circuit. This characteristic is primarily a function of the applied frequency and the solution/cell properties being dependent on the solution conductivity and dielectric permittivity at any given concentration. Small changes in sample dielectric permittivity in the presence of a large background conductivity are shown to be significant. The apparatus described can provide fixed or swept frequency conductivity measurements in the range 1 kHz to 2.25 MHz with a lower conductivity sensitivity of 0.9 x 10(-3) Scm-1. Bulk impedimetric characteristics of cell suspensions are derived by a two stage measurement.

Pneumonia due to Branhamella catarrhalis.

In 12 of 451 patients diagnosed as having pneumonia in a single hospital over 18 months the causative organism appeared to be Branhamella catarrhalis.

Imipenem-cilastatin in the treatment of respiratory infections in patients with chronic airways obstruction.

Chest infections with organisms resistant to conventional antibiotics are common in patients with chronic lung disease. We have studied the use of imipenem in 40 (28 M 12 F) patients admitted for treatment of chest infections. Patients were treated with imipenem 0.5 g four times daily by intravenous infusion for 6.3 +/- 1.6 (S.D.) days. Forty-six respiratory pathogens were cultured from 36 patients including 18 Haemophilus influenzae, 6 H. parainfluenzae, 6 Streptococcus pneumoniae, 8 Pseudomonas aeruginosa, and 6 Branhamella catarrhalis. Forty-three of the 46 isolates were sensitive to imipenem, 28 to ampicillin, 33 to tetracycline and 35 to cotrimoxazole. Thirty-eight of the 40 patients improved clinically, and 34 of the 36 patients with positive sputum culture had no pathogens in their sputum after treatment. Twenty patients developed minor phlebitis at the infusion site but there were few other side effects. Imipenem may prove useful in the treatment of chest infections, particularly when the organism is resistant to conventional antibiotics.

Characterisation of Branhamella catarrhalis and differentiation from Neisseria species in a diagnostic laboratory.

To distinguish Branhamella catarrhalis from Neisseria species a study of 140 strains was made on simple laboratory media, with particular reference to deoxyribonuclease (DNase) production, superoxol reaction, and growth characteristics. All 97 clinical isolates of B catarrhalis (58 of which were beta-lactamase positive) and eight strains of B catarrhalis from the National Collection of Type Cultures were DNase positive and superoxol positive. None grew on modified New York City medium, modified Thayer Martin medium, MacConkey agar, crystal violet blood agar, nor under anaerobic conditions. Of the 16 different non-pathogenic Neisseria species tested, all were DNase negative, eight (50%) were superoxol reaction negative, and 13 (81%) grew on crystal violet blood agar. Using simple laboratory media, DNase, and superoxol tests, it was possible to identify B catarrhalis and to distingish it from pathogenic and non-pathogenic Neisseria species.

Pneumococcal serotypes in sputum isolates during acute respiratory illness in Edinburgh.

During the years 1978-83 serotyping was carried out on all sputum isolates of pneumococci obtained from patients in the chest wards of the City Hospital, Edinburgh. In 402 patients with acute respiratory illness the peak isolation rates occurred from January to April, and the serotype distribution was similar to that seen in previous UK studies, the commonest types being 3, 6, 9, 19, 23, and 8. The overall mortality rate was 8.7%, the serotype distribution in fatal cases reflecting the distribution of the whole group. The presence of mixed infection, predominantly with Haemophilus influenzae, was associated with a lower mortality rate of 3.5%. Nearly all patients (92%) were either elderly or had a chronic underlying disease and only one death occurred in a patient under 70 years who had no pre-existing disease. Of the pneumococcal serotypes isolated from the 292 patients with chronic chest disease, 82% are included in the new 23 valent pneumococcal vaccine and the efficacy of this needs to be assessed further in high risk patients.