[Nebulization of emergency medications in the south German rescue service]. / Vernebelung von Notfallmedikamenten im süddeutschen Rettungsdienst.

BACKGROUND: In German emergency rescue services, inhalation treatment is routinely carried out by qualified health personnel. Standard operating procedures (SOP) for nebulization are neither uniform throughout Germany nor available in all federal states. Standardized recommendations with respect to which nebulizer type should be used are missing. The aerosol output as well as the drug deposition rates of jet and mesh nebulizers, however, differ considerably. Mesh devices can achieve a threefold higher lung deposition. Their use in emergency departments has also been shown to be associated with a better patient outcome when compared to jet nebulizers. OBJECTIVE: This survey was designed to evaluate the type of nebulizer used in the south German rescue services. Special attention was paid to the influence of existing SOP on the decision to perform nebulization during emergency treatment. MATERIAL AND METHODS: A total of 4800 emergency paramedics working in Baden-Württemberg, Bavaria and Rhineland-Palatinate received a questionnaire with a total of 17 questions on the implementation of drug nebulization in the daily practice. RESULTS: Despite the existence of more efficient nebulizer types, the jet nebulizer was by far the most frequently used nebulizer in the south German rescue services. The deposition rates of both the jet and mesh nebulizers were considerably overestimated by most respondents; however, 77.5% of all respondents could not give any information about the deposition rates of the mesh nebulizer. Only two thirds of all respondents carried out nebulization treatment on the basis of SOP. The implementation of SOP, however, was pivotal to the application of nebulization during emergencies. If SOP were in place,76.9% of the responders used aerosol treatment compared to 23.1% when there were none. The perceived safety when using nebulization during emergencies was also significantly higher (p = 0.013) when SOP were implemented. CONCLUSION: The exclusive use of mesh nebulizers could standardize the treatment of emergency patients in the south German rescue services. The use of mesh devices might possibly improve patient outcomes, even if clinical studies are still lacking. Nebulizer treatment differs between the federal states. A comprehensive implementation of SOP for nebulization treatment might support this process and could increase the application frequency and the perceived safety of nebulization during emergencies. A better training of paramedic personnel could improve the knowledge of aerosols as a treatment option for emergency patients and help to classify the advantages and disadvantages of the different aerosol generators available.

[Relevant incidental findings and iatrogenic injuries : A retrospective analysis of 1165 resuscitation room patients]. / Relevante Zufallsbefunde und iatrogene Verletzungen : Eine retrospektive Analyse von 1165 Schockraumpatienten.

BACKGROUND: Whole-body computed tomography (CT) is increasingly being used as the diagnostic modality of choice in patients admitted to the resuscitation room. Beyond findings related to the suspected diagnosis it often additionally reveals incidental findings. The aim of this investigation was the evaluation of these findings in patients admitted via the emergency room after suffering potential major trauma or life-threatening medical conditions. Furthermore, the number of iatrogenic injuries as well as misplaced catheters and endotracheal tubes was investigated. METHODS: All patients admitted from 1 February 2012 to 31 January 2014 via the resuscitation area of the Mannheim University Medical Center, a tertiary care hospital and level 1 trauma center, were included in this study if they had undergone a whole-body CT scan at admission. Data from 1362 patients were collected retrospectively and 197 patients were excluded because of missing data so that the final cohort consisted of 1165 patients (1038 trauma and 127 internal neurological patients). Reports from the whole-body CT scans were screened for incidental findings. These findings were then classified as either clinically relevant or not. Furthermore, the reports were checked for iatrogenic injuries as well as misplaced catheters and endotracheal tubes. RESULTS: A total of 465 incidental findings were reported in 293 patients (25.1%) of the final cohort. In the synopsis of the radiological and clinical findings, 72 were rated as clinically relevant. In one patient two relevant incidental findings were reported and one patient presented with three incidental findings. In total, relevant incidental findings could be detected in 5.8% of the study patients (68/1165). In the discharge letters and/or the radiological report 16.2% of the incidental findings rated as clinically relevant were reported to be previously known, 66.2% were reported to be unknown and 17.6% could not be unequivocally classified as known or unknown due to missing references in the discharge letters. The group of internal neurological patients were clearly older than the trauma patients (61.6 years vs. 45.5 years). The rate of relevant incidental findings in the internal neurological group was more than twice as high as in the trauma group (11.0% vs. 5.2%); however, in the relatively young trauma group 1 in 20 patients showed an incidental finding classified as clinically relevant. In 43 (3.7%) patients a total of 46 iatrogenic injuries or misplaced catheters were reported. The most common finding was a too deeply placed endotracheal tube and five transurethral catheters placed in the emergency room were found to be blocked within the urethra. CONCLUSION: In addition to the main diagnosis, clinically relevant incidental findings were reported in nearly 25% of whole-body CT scans of patients admitted to the resuscitation room. Approximately 6% of patients had incidental findings rated as clinically relevant. In the internal neurological group of patients the rate of incidental findings was doubled compared to the trauma group; however, the latter were significantly younger. Whole-body CT was also useful for diagnosing iatrogenic injuries and misplaced catheters in approximately 4% of the study patients.

[Preclinical fibrinolysis in patients with ST-segment elevation myocardial infarction in a rural region]. / Präklinische Fibrinolyse bei Patienten mit ST-Hebungsinfarkt in einer ländlich geprägten Region.

BACKGROUND: In the current guidelines for the treatment of patients with ST-segment elevation myocardial infarction (STEMI), the European Society of Cardiology (ESC) recommends preclinical fibrinolysis as a reperfusion therapy if, due to long transportation times, no cardiac catheterisation is available within 90-120 min. However, there is little remaining in-depth expertise in this method because fibrinolysis is presently only rarely indicated. METHODS: In a rural area in southwestern Germany, where an emergency primary percutaneous coronary intervention was not routinely available within 90-120 min, 156 STEMI patients underwent fibrinolysis with the plasminogen activator reteplase, performed by trained emergency physicians. The practicality of the treatment, as well as complications and the mortality of the patients in the preclinical phase until arrival at the hospital, were retrospectively studied. RESULTS: The mean time from onset of the symptoms to first medical contact was 114 ± 116 min. The mean interval to the start of fibrinolysis of 13.5 ± 6.4 min was within the 30 min mandated by the ESC. Patients with inferior STEMI represented the largest subgroup. Occurring in 39 cases (25 %), complications due to infarction were relatively common during the prehospital phase, including 15 cases (9.6 %) of cardiogenic shock, but in all cases the complications were manageable. No patient died before arrival at the hospital. As lysis-associated adverse effects, merely two uncomplicated mucosal haemorrhages and one case of mild allergic skin reactions were seen. CONCLUSION: In emergency situations with long transportation times to the nearest suitable cardiac catheterisation laboratory, preclinical fibrinolysis in STEMI still represents a workable method. Success of this strategy requires particularly strong training of the emergency physicians in ECG and lysis therapy, and co-operation with nearby cardiac centres.

Inactivation of the LOX-1 pathway promotes the Golgi apparatus during cell differentiation of mural granulosa cells.

In female mammals, granulosa cells of the ovarian follicle differentiate into the corpus luteum after ovulation of the pregnable oocyte into the fallopian tube. During these differentiation processes several morphological alterations have to occur and the molecular basis is not fully understood. As an endpoint estradiol production from granulosa cells has to switch off in favor for progesterone production from the proceeding corpus luteum to sustain the developing embryo. Previously, we demonstrated that the multiligand receptor LOX-1 plays a critical role in steroid hormone synthesis of granulosa cells via intracellular calcium release from endoplasmic (ER)-dependent and ER-independent calcium pools. In the present study, we show that inhibition of LOX-1 leads to a rearrangement of ceramide from the basal membrane toward the Golgi apparatus. This activity is accomplished by a calcium-dependent phosphorylation of aromatase, the key step in estradiol production. Phosphorylated aromatase increased estradiol production in a dose-dependent manner. Our data indicate that the ceramide cascade is essential for proper granulosa cell function and ceramide redistribution serves as a first step in order to proceed with the prosperous differentiation into a corpus luteum.

LOX-1 regulates estrogenesis via intracellular calcium release from bovine granulosa cells.

Estradiol produced by ovarian granulosa cells triggers the luteinizing hormone surge which in turn initiates ovulation in female mammals. Disturbances in estradiol production from granulosa cells are a major reason for reproductive dysfunctions in dairy cows. Endogenous estradiol production might be altered by reactive oxygen species (ROS) such as oxidized low-density lipoprotein (ox-LDL). Inhibition of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), a receptor of ox-LDL, leads to increased estrogenesis in granulosa cells. This activity is mediated by calcium release from endoplasmic reticulum (ER)-dependent and ER-independent calcium pools. Inhibition of the LOX-1 signal transduction pathway is followed by mitochondrial alterations. The membrane potential ΔΨ increases and the ROS production decreases in mitochondria after blocking LOX-1. Our data indicate that blocking the LOX-1 receptor signal pathway might be a promising way to improve steroid hormone concentrations in metabolically highly active female mammals and, therefore, to defend against reproductive dysfunctions in humans and animals.

Postpartum levels of 8-iso-prostaglandin F2α in plasma and milk phospholipid fractions as biomarker of oxidative stress in first-lactating dairy cows.

F2-isoprostanes such as 8-iso-prostaglandin F2 (8-iso-PGF2α) are formed by free radical-catalyzed mechanisms from membrane phospholipids and from low density lipoproteins through peroxidation of arachidonic acid. Esterified 8-iso-PGF2α is cleaved by phospholipases, circulates in blood and is excreted as putatively harmful oxidatively modified lipid via the kidney into urine. In this study we demonstrate that 8-iso-PGF2α concentrations in plasma samples from heifers are higher (p<0.005) compared to those from first-lactating dairy cows at 71 days postpartum. Furthermore, plasma 8-iso-PGF2α concentrations vary with ovarian activity and differ in response to luteolytic initiation as well as activation of the hypothalamic-pituitary-gonadal axis between heifers and first-lactating cows. Sustainable concentrations of 8-iso-PGF2α (50-150 pg/ml) are detectable in the phospholipid fraction of milk, suggesting milk as an additional excretion route for 8-isoprostanes. Plasma levels largely paralleled levels in milk (p<0.001). Plasma phospholipid 8-iso-PGF2α concentrations in cyclic cows decreased (p<0.05) from day 38 to day 71 postpartum, whereas milk phospholipid 8-iso-PGF2α rather increased (p<0.05). Cyclic cows tend to have higher 8-isoprostane levels compared to acyclic animals. In contrast to lipohydroperoxides, concentration of 8-iso-PGF2α were not correlated with milk yield (p>0.05). Our data indicate 8-iso-PGF2α may be a novel biomarker of oxidative stress in dairy cow, which is detectable in blood as well as in milk.

[Spectrum of missions for a rescue helicopter. Changes in a south German urban area over the last 25 years]. / Einsatzspektrum eines Rettungshubschraubers. Veränderung in einem süddeutschen Ballungsgebiet in den letzten 25 Jahren.

BACKGROUND: Over the past decade the number of air rescue missions has increased continuously. The reasons for this are still discussed at great length. In addition to the demographic changes to becoming an increasingly older and sicker society, the political reform in the healthcare system with a simultaneous reduction and concentration of hospitals and formation of centers is also under discussion. AIM: The key questions to be answered are, therefore, can the increasing demands on the emergency physician really be explained by an increasing number of severely ill and injured patients? Is a proportion of the missions really not indicated because they do not involve immediately life-threatening "acute medical" emergencies and are caused by a lack of alternative paramedical service infrastructures, unavailability of a general practitioner or the only temporary availability of medical on-call standby services? MATERIAL AND METHODS: This study therefore analyzed the alterations of a possible change with respect to the spectrum of missions and the utilization of air rescue services in the metropolitan area of Stuttgart. All primary missions of the rescue helicopter (RTH) "Christoph 41" (based in Leonberg) from 2006 to 2011 were included in the study and compared to the data from 1987 to 1992. RESULTS: The indications for missions and the spectrum of patients for the RTH Christoph 41 have changed over the last 25 years. The proportion of emergency trauma cases has significantly decreased and missions for non-trauma cases have increased. The proportion of patients with life-threatening conditions has increased. Despite the change in the spectrum of missions, emergency physicians are confronted with a greater number of patients with life-threatening conditions than 25 years ago. The patients treated were on average clearly older than the patients who were treated by the air rescue service 25 years ago. CONCLUSION: The changes in the spectrum of missions up to more emergencies involving non-trauma patients and older patients must be taken into consideration during training and advanced training.

Fuel feeds function: Energy balance and bovine peripheral blood mononuclear cell activation.

A general phenomenon in peripartum mammals is the breakdown of (acquired) immunity. The incidence of parasite load, disease and inflammation often rise during the specific energetically demanding time of pregnancy and lactation. In this period, blood leukocytes display decreased DNA synthesis in response to mitogens in vitro. Leukocyte activation, the phase of the cell cycle preceding the DNA synthetic phase has hardly been investigated, but the few studies suggest that leukocyte activation may also be impaired by the limited energy/nutrient availability. Leukocyte activation is characterized by manifold processes, thus, we used the cellular oxygen consumption rate (OCR) as a measure of ATP turnover to support all these processes. We hypothesized that the activation of peripheral blood mononuclear cells (PBMC) - in terms of oxygen consumed over basal levels after in vitro stimulation - is altered by energy balance around parturition. We studied peripartum high-yielding dairy cows because they undergo substantial fluctuations in energy intake, energy output and body fat mass. We established a fluorescence-based test strategy allowing for long-term (≥24h) quantification of O(2)-consumption and studied the peripartum period from 5 weeks ante partum to 5 weeks postpartum. In addition, we determined cellular lactate production, DNA/RNA synthesis and cell size and zoo-technical parameters such as animal energy intake and milk yield were assessed, as well as selected plasma parameters, e.g. glucose concentration. The basal OCR of PBMC from pregnant, non-lactating cows (n=6, -5 weeks ante partum) was 1.19±0.15 nmol min(-1) (10(7)cells)(-1) and increased to maximum levels of 2.54±0.49 nmol min(-1) (10(7)cells)(-1) in phytohemagglutinin (PHA)-stimulated PBMC. The basal OCR did not change over the peripartum period. Whereas the activation indices, herein defined as the PHA-induced 24h-increase of OCR above baseline, amounted to 1.1±0.3, 4.2±0.3, 4.1±1.1, 2.1±0.3, and 2.7±0.5 at weeks -5, -1, +1, +2, and +5 relative to parturition, respectively. Because the activation index was positively correlated to plasma glucose levels and to energy balance during late pregnancy (week -5/week -1) and transition to lactation (week -1/week +2), we conclude that PBMC activation is modulated by energy/nutrient availability. In future studies, the activation index should aid the identification of causal mechanisms of disparity in PBMC activation, such as attenuated ion transport or macromolecule synthesis.

Perilipin, C/EBPα, and C/EBPß mRNA abundance in longissimus muscle and different adipose tissues of Holstein and Charolais cattle.

Possible regulators of intramuscular fat deposition were investigated in longissimus muscle (LM) and adipose tissues of 18months old Holstein and Charolais bulls. The mRNA abundance of perilipin and transcription factors C/EBPα and C/EBPß was analyzed by real-time RT-PCR. Carcass traits and marbling traits were recorded and relationships among adipogenic genes and tissue traits were determined. Charolais cattle were heavier (P<0.001) and had less body fat (P<0.001). Holstein bulls accumulated more fat in the LM (P=0.02), but the number and size of marbling flecks did not differ (P>0.7) between breeds. Perilipin, C/EBPα, and C/EBPß mRNA abundance was influenced by tissue but not by breed. Relationships between mRNA abundance and marbling traits could not be confirmed, however relationships among adipogenic genes. The transcriptional activity of adipogenic genes in LM suggests that intramuscular adipose tissue is still developing and differentiation still occurs.

Nutritional level and energetic source are determinants of elevated circulatory lipohydroperoxide concentration.

Dietary energetic impact on oxidative stress is incompletely understood. Therefore, effects of diets on oxidative stress were studied using a crossover block design. In Expt 1, intake of metabolizable energy (ME) was restricted or ad libitum. In Expt 2, isoenergetic and isonitrogenic diets were fed, replacing carbohydrate energy by energy of fatty acids. Circulatory lipohydroperoxides (LOOH), markers of acute oxidative stress, were expressed absolutely and in terms of cholesterol or TAG levels. In Expt 1, plasma (jugularis vein) LOOH was assayed in combination with whole-body oxidative metabolism using gas exchange and heart rate (HR) during feeding periods and at rest. In Expt 2, LOOH was assayed in plasma from portal and a large udder vein and a mesenteric artery. In Expt 1, intake increased VO2, HR and LOOH following overnight fast with higher values (P < 0.05) when feeding ME ad libitum. Intake of ME ad libitum (3 weeks) increased cardiac protein of cytochrome oxidase and endothelial-type nitric oxide synthase (P < 0.05), indicating adaptation of the heart to higher activity. Transient HR responses evoked by an antidiabetic drug (levcromakalim) revealed a linear positive correlation with relative LOOH (r2 0.79), supporting the relationship between oxidative metabolic rate and lipoperoxidation. Evidence for exogenous lipids as LOOH source provided the vessel-specific rise in LOOH through replacing carbohydrate ME by lipid ME (Expt 2). Thus, dietary energy level and energetic source are important for circulatory LOOH with a role of vascular activity in production of oxidant.

Diagnostic Performance of Contrast Enhanced Pulmonary Computed Tomography Angiography for the Detection of Angioinvasive Pulmonary Aspergillosis in Immunocompromised Patients.

Invasive pulmonary aspergillosis (IPA) is one of the major complications in immunocompromised patients. The mainstay of diagnostic imaging is non-enhanced chest-computed-tomography (CT), for which various non-specific signs for IPA have been described. However, contrast-enhanced CT pulmonary angiography (CTPA) has shown promising results, as the vessel occlusion sign (VOS) seems to be more sensitive and specific for IPA in hematologic patients. The aim of this study was to evaluate the diagnostic accuracy of CTPA in a larger cohort including non-hematologic immunocompromised patients. CTPA studies of 78 consecutive immunocompromised patients with proven/probable IPA were analyzed. 45 immunocompromised patients without IPA served as a control group. Diagnostic performance of CTPA-detected VOS and of radiological signs that do not require contrast-media were analyzed. Of 12 evaluable radiological signs, five were found to be significantly associated with IPA. The VOS showed the highest diagnostic performance with a sensitivity of 0.94, specificity of 0.71 and a diagnostic odds-ratio of 36.8. Regression analysis revealed the two strongest independent radiological predictors for IPA to be the VOS and the halo sign. The VOS is highly suggestive for IPA in immunocompromised patients in general. Thus, contrast-enhanced CTPA superior over non-contrast_enhanced chest-CT in patients with suspected IPA.

Comparative study of cell cycle kinetics and induction of apoptosis or necrosis after exposure of human Mono Mac 6 cells to radiofrequency radiation.

The possible harmful effects of radiofrequency electromagnetic fields (RF EMFs) are controversial. We have used human Mono Mac 6 cells to investigate the influence of RF EMFs in vitro on cell cycle alterations and BrdU uptake, as well as the induction of apoptosis and necrosis in human Mono Mac 6 cells, using flow cytometry after exposure to a 1,800 MHz, 2 W/kg specific absorption rate (SAR), GSM-DTX signal for 12 h. No statistically significant differences in the induction of apoptosis or necrosis, cell cycle kinetics, or BrdU uptake were detected after RF EMF exposure compared to sham or incubator controls. However, in the positive control cells treated with gliotoxin and PMA (phorbol 12 myristate-13 acetate), a significant increase in apoptotic and necrotic cells was seen. Cell cycle analysis or BrdU incorporation for 72 h showed no differences between RF EMF- or sham-exposed cells, whereas PMA treatment induced a significant accumulation of cells in G(0)/G(1)-phase and a reduction in S-phase cells. RF EMF radiation did not induce cell cycle alterations or changes in BrdU incorporation or induce apoptosis and necrosis in Mono Mac 6 cells under the exposure conditions used.

Endocrine, morphological, and cytological effects of a depot GnRH agonist in bovine.

The present study was conducted to assess effects of the gonadotropin-releasing hormone agonist (GnRHa) triptorelin in dairy heifers. The peptide was released from a commercial 4-week depot formulation (Decapeptyl Depot) administered at animals' estrus (day 0). First experiment (EXP I, n=5), which was aimed to explore the availability of peptide, detected a maximum of triptorelin concentration between day 2 and 5 after depot injection, and the peptide remained detectable by RIA in peripheral blood for about 3 weeks. In further experiments, the peptide release was terminated on day 9 (EXP II, n=16) or day 21 (EXP III, n=47). Treatment effects were studied on follicular development, the characteristics of cumulus-oocyte complexes (COCs) (EXP II; EXP IIIa) and secretions of LH and progesterone (EXP IIIb). Results showed that the occurrence of the pre-ovulatory LH surge was more uniform in treated heifers than that in controls. The duration of ovulation periods was similar amongst the heifers of EXP II, but more compact amongst those of EXP III each compared with the respective controls. Post-ovulatory, the number of LH pulses was significantly reduced by treatment, whereas both basal LH and progesterone concentrations were elevated on a few days. Follicular growth was reduced only by the prolonged influence of the GnRHa. There were increased proportions of both degenerated COCs and immature oocytes from small follicles (<3mm in diameter), and meiotic configuration and quality of oocytes isolated from follicles 3-5mm were changed after the prolonged, 21-day treatment. These results indicate that a continuous influence of a GnRHa over more than 1 week may increasingly impair the development of bovine follicles and oocytes. This may have some significance for the development of novel GnRH-based techniques in regulating the reproductive function in cattle.

Polar phospholipids from bovine endogenously oxidized low density lipoprotein interfere with follicular thecal function.

The role of endogenously oxidized low density lipoprotein (oxLDL) in follicular steroidogenic regulation is unknown. Information may be important in order to elucidate ovulatory dysregulation in disordered lipid metabolism. To obtain specific data, we studied the effect of polar phospholipids (PL) isolated from oxLDL with different endogenous levels of lipohydroperoxides (LHP) on the thecal expression of mRNA encoding steroidogenic enzymes and cyclooxygenase 2 (COX-2), and on the thecal production of superoxide and progesterone. Large (preovulatory) bovine follicles were used and analyses of thecal fragments from single follicles were performed by radioimmunoassays, chemiluminescence assays and quantitative RT-PCR. Basal concentration of mRNA for several lipoprotein receptors exceeded by about 10-times the basal level of mRNA encoding steroidogenic enzymes, suggesting that preovulatory theca receptors may favour uptake of oxLDL. PL (5-11 pmol phosphorus/ml) decreased (up to 0.5-times the control) progesterone synthesis, production of superoxide and levels of P450 cholesterol side chain cleavage (P450 scc), 3beta-hydroxysteroid dehydrogenase and COX-2 mRNA. Abundance of COX-2 transcripts in thecal tissue incubated with forskolin depended on the progesterone/17beta-oestradiol ratio of the follicle fluid, i.e. the previous microenvironment in vivo. PL effects were mimicked by the platelet-activating factor (PAF). WEB 2086, a PAF receptor blocker, did not always abolish these responses, suggesting that the effects were not mediated solely by this receptor. PAF interfered dose-dependently with LH-induced responses, indicating interference with LH signalling. PL from mildly oxidized LDL (0.5 nmol/ml LHP) tended to exert greater effects than PL from oxLDL containing 1.5 nmol/ml LHP. In consideration of the known physiologic role of progesterone, COX-2 and possibly superoxide, these results provide evidence for a potential of PL from oxLDL to induce ovulatory dysregulation and suggest that the extent of the LDL oxidation seems to be important for interfering with thecal responses to the preovulatory LH surge.

Bovine blastocyst development rate in vitro is influenced by selection of oocytes by brillant cresyl blue staining before IVM as indicator for glucose-6-phosphate dehydrogenase activity.

The aim of this present study was to increase the efficiency of blastocyst production from cows after in vitro maturation/fertilization (IVM/IVF) by oocyte selection before maturation. Oocytes were selected on the basis of brillant cresyl blue (BCB) staining, used to indicate glucose-6-phosphate dehydrogenase (G6PDH) activity. To re-valuate the hypothesis that growing oocytes are expected to have a high level of active G6PDH, while mature oocytes have low G6PDH activity, cumulus oocyte complexes (COCs) were recovered from slaughterhouse ovaries by slicing the surface of the ovary. Only oocytes with a compact cumulus investment were used. Oocytes were placed into three groups: (1) control--placed immediately into culture; (2) holding control--COCs kept in PBS containing 0.4% BSA for 90 min before placement into culture; and (3) treatment--incubation with BCB for 90 min before culture. Treated oocytes were then divided into BCB- (colorless cytoplasm, increased G6PDH) and BCB+ (colored cytoplasm, low G6PDH) on their ability to metabolize the stain. Activity of G6PDH was determined via measurement of NADP reduction induced by G6P as substrate oxidized by G6PDH in the cytosol of control, BCB- and BCB+ groups; G6PDH activity was significant higher in BCB- COCs than in control and BCB+ COCs. After IVM, oocytes were fertilized in vitro. Embryos were cultured to day 8. The rate of maturation to metaphase II was significantly higher for control and BCB+ oocytes than for BCB- oocytes. The BCB+ oocytes yielded a significantly higher proportion of blastocysts (34.1%) than did control or holding control oocytes (18.3 and 19.2%); and both controls and BCB+ oocytes had significantly higher blastocyst development than did BCB- oocytes (3.9%). These results show that the staining of bovine cumulus oocyte complexes with BCB before in vitro maturation may be used to select developmentally competent oocytes for IVF. In addition, G6PDH activity may be useful as a marker for oocyte quality in future studies on factors affecting developmental competence.

Modifications in cell cycle kinetics and in expression of G1 phase-regulating proteins in human amniotic cells after exposure to electromagnetic fields and ionizing radiation.

Low-frequency electromagnetic fields are suspected of being involved in carcinogenesis, particularly in processes that could be related to cancer promotion. Because development of cancer is associated with deregulated cell growth and we previously observed a magnetic field-induced decrease in DNA synthesis [Lange et al. (2002) Alterations in the cell cycle and in the protein level of cyclin D1p, 21CIP1, and p16INK4a after exposure to 50 HZ. MF in human cells. Radiat. Environ. Biophys.41, 131], this study aims to document the influence of 50 Hz, 1 mT magnetic fields (MF), with or without initial gamma-ionizing radiation (IR), on the following cell proliferation-relevant parameters in human amniotic fluid cells (AFC): cell cycle distribution, expression of the G1 phase-regulating proteins Cdk4, cyclin D1, p21CIP1 and p16INK4a, and Cdk4 activity. While IR induced a G1 delay and a dose-dependent G2 arrest, no discernible changes in cell cycle kinetics were observed due to MF exposure. However, a significant decrease in the protein expression of cyclin D1 and an increase in p21CIP1- and p16INK4a-expression could be detected after exposure to MF alone. IR-exposure caused an augmentation of p21CIP1- and p16INK4a- levels as well, but did not alter cyclin D1 expression. A slight diminution of Cdk4 activity was noticed after MF exposure only, indicating that Cdk4 appears not to act as a mediator of MF- or IR-induced changes in the cell cycle of AFC cells. Co-exposure to MF/IR affected neither cell cycle distribution nor protein expression or kinase activity additionally or synergistically, and therefore MF seems not to modify the mutagenic potency of IR.

Detection and functional characterisation of the transcription factor peroxisome proliferator-activated receptor gamma in lutein cells.

A prominent functional change during differentiation of lutein cells from follicular thecal and granulosa cells is an enhanced production and secretion of progestins. The regulation of this process is not fully understood but may be associated with the expression of transcription factors which activate genes, products of which are involved in pathways of the cholesterol and lipid metabolism. As peroxisome proliferator-activated receptors (PPARs) play a role in both pathways, we were interested in the expression of PPARgamma, a PPAR form which is involved in adipogenic differentiation. First, we were able to show the expression of PPARgamma in bovine lutein cells (day 12 of the ovarian cycle) at the mRNA and protein level by imaging, flow cytometry and blot analysis, and secondly a role of PPARgamma in the secretion of progesterone. The cells (24 h culture) responded dose dependently by increasing progesterone secretion (up to 1.5-fold of the basal level) to an endogenous ligand of PPARgamma, 15-deoxy-delta12,14 prostaglandin J2 (15-dPGJ2) and to the thiazolidinedione ciglitizone. Aurintricarboxylic acid (ATA) was found to reduce the intracellular PPARgamma level and to promote cell cycle progress, indicating that ATA can be used as a tool for experimental changes of PPARgamma proteins in intact cells and for studying the physiological consequences. The ATA-mediated decrease of PPARgamma was accompanied by reduced progesterone production and a progression of the cell cycle, suggesting a function of PPARgamma in both processes. The response to ATA was abrogated by a high dose (>490 nM) of 15-dPGJ2, suggesting that 15-dPGJ2 exerts its effect on steroidogenic activity via PPARgamma and that the 15-dPGJ2-PPARgamma system plays a role in the maintenance of a differentiated quiescent stage in lutein cells.

Influence of the mycotoxins alpha- and beta-zearalenol and deoxynivalenol on the cell cycle of cultured porcine endometrial cells.

The present study investigated the effects of the mycotoxins alpha-zearalenol (alpha-ZOL) and beta-zearalenol (beta-ZOL) at concentrations of 7.5, 15, and 30 microM, and deoxynivalenol (DON) at concentrations of 0.94, 1.88, and 3.76 microM on cell cycle distribution (propidium iodide, PI staining) in combination with the proliferating cell nuclear antigen (PCNA) by flow cytometry. The viability of porcine uterine cells was not impaired at 30 microM alpha-ZOL, whereas beta-ZOL at this concentration and 3.76 microM DON significantly decreased cell number. Some cells showed ultrastructural features of cell death indicated by swollen mitochondria, disrupted cell membranes, and many vacuoles. After 24 and 48h of exposure to alpha-ZOL (7.5, 15, or 30 microM), the cell cycle distribution was still comparable to the control groups. An anti-proliferative effect of beta-ZOL and DON was detected by a significant reduction in the S-phase together with arrest of cells in the G(0)/G(1)-phase. The results show that beta-ZOL (7.5, 15, or 30 microM) and DON (0.94, 1.88, or 3.76 microM) control the progression of cells through the cycle by decreasing S-phase and arresting cells in the G(0)/G(1)-phase of the cell cycle. A significant decrease in the expression of the proliferation marker PCNA amounts indicates that beta-ZOL and DON disengaged cells from active cycling. We confirm that alpha-ZOL possesses a relative binding affinity to porcine uterine cytoplasmic estrogen receptor.

Influence of organochlorine pesticides on transmembrane potential, oxidative activity, and ATP-induced calcium release in cultured bovine oviductal cells.

The present study investigated the effects of the pesticides DDT, MXC, and gammaHCH on transmembrane potential, oxidative activity, cytotoxicity and ATP-induced intracellular Ca2+ release in cultured bovine oviductal cells. Transmembrane potential, oxidative activity, and cytotoxicity were assessed using the fluorescent dyes bis-oxonol, dihydrorhodamine 123, and propidium iodide (PI), respectively, and measured spectrofluorometrically in a microplate reader. The cultured cells were loaded with Ca2+-sensitive fluorochrome fura-2-AM, and cytosolic free calcium ([Ca2+]i) was monitored by a microscope image analysis system. A dose-dependent increase in depolarization and changes of oxidative activity were observed over a concentration range of 8 to 128 microM DDT and MXC compared to nonexposed controls. At a concentration of 16 microM DDT or MXC, the oxidative activity and depolarization of cells were significantly enhanced compared to controls, but most of the cells were intact as indicated by the fact that PI-staining was not significantly increased. Trypan-blue staining indicated that the viability of oviductal cells decreased significantly when exposed to concentrations of 64 and 128 microM DDT or MXC. ATP-mediated enhancement of [Ca2+]i in cells was almost completely inhibited after incubation with 128 microM DDT for 3 h at 37 degrees C. This response was reduced to approximately 50% after incubation of the cells with MXC at 128 microM; lindane did not significantly interfere with the above physiologic parameters.

Fluorometric detection of platelet activating factor receptor in cultured oviductal epithelial and stromal cells and endometrial stromal cells from bovine at different stages of the oestrous cycle and early pregnancy.

During the oestrous cycle and early pregnancy, the oviduct and uterus undergo a variety of morphological and physiological modifications in which the platelet activating factor receptor (PAF-R) plays an important role. PAF-R levels were quantified in bovine oviductal epithelial and stromal cells and endometrial stromal cells at days 2 to 4, 12, and 20 of the estrous cycle and during early pregnancy. Cells were grown in vitro and their intracellular PAF-R concentration was measured by flow cytometry using a polyclonal anti-PAF-R antibody system. A significant increase (P < 0.05) in the portion of PAF-R-positive oviductal epithelial and stromal cells was detected in both non-pregnant and pregnant cattle on days 2 to 4 in comparison to day 12 and 20. In endometrial stromal cells derived from day 20 pregnant bovine, a significant increase (P < 0.05) in PAF-R staining was observed in comparison to the day 20 non-pregnant and days 2 to 4 or 12 pregnant and non-pregnant animals. The PAF-R was detected in oviductal cells by using immunoblotting and immuno-gold postembedding method. Positive binding of the anti-PAF-R antibody was found on the cell membrane and in the cytoplasm. We concluded that the increased PAF-R concentration measured in cultured oviductal epithelial and stromal cells of cyclic and pregnant heifers on days 2 to 4 was hormonally regulated. The increased PAF-R in endometrial stromal cells on day 20 of pregnant heifers was a pregnancy-specific effect and may mediate a local increase in endometrial vascular permeability known to precede the implantation.