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.

Role of the platelet-activating factor and its receptor in the proliferative regulation of bovine ovarian granulosa cells.

UNLABELLED: Proliferation of granulosa cells and their withdrawal from the cell cycle may regulate follicular ovulation. Antagonists of platelet-activating factor (PAF) and its receptor (PAFr) inhibit follicle rupture. OBJECTIVES: Thus, PAF and PAFr may be involved in proliferative regulation of granulosa cells; however, expression of PAFr in these cells is unknown. MATERIALS AND METHODS: The aim of this study was to investigate the presence of PAFr and the effect of PAF on proliferation of cultured bovine granulosa cells using real-time polymerase chain reaction to assay steady-state level of mRNA, immunocytochemistry to quantify PAFr protein and proliferating cell nuclear antigen protein by flow cytometry. RESULTS: We found that granulosa cells express PAFr transcripts and protein. PAF presence did not change the concentration of PAFr mRNA or PAFr protein. Granulosa cells responded to PAF doses of 10 and 50 nm with increasing proportions of cells entering G0/G1 phase, as well as a significant expansion of total cell numbers. Rise in G0/G1-phase cells was accompanied by a decline in proliferating cell nuclear antigen protein expression, and these effects could be suspended by simultaneous PAFr blockage. The results provide clear evidence for expression of PAFr in bovine granulosa cells and its functional involvement in PAF/PAFr-mediated stimulation of cell recruitment. CONCLUSIONS: PAF antagonists are suggested to disturb this regulative activity of PAF and to contribute in this way to blockage of ovulation.

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.

Short communication: hydroperoxides in circulating lipids from dairy cows: implications for bioactivity of endogenous-oxidized lipids.

This study was conducted to investigate the potential for increased oxidative stress of high- vs. average-producing dairy cows. Two experiments were performed using 11 and 13 Holstein cows (53 +/- 2 d postpartum). Lipohydroperoxides (LHP) were determined in serum lipids (experiment 1) and low-density lipoprotein (experiment 2) via oxidation of ferrous to ferric ions through LHP using thiocyanate as chromogen. In experiment 1, differing milk yield and milk energy output corresponded to different concentrations of LHP. In experiment 2, analysis of regression resulted in a significant relationship between milk yield and LHP. Phospholipids isolated from lipids with 6.5 microM of LHP evoked in monocytic cells a transient increase in superoxide formation, indicating inflammatory potential. The results show that high milk productivity can associate with oxidative stress indicated by oxidative modifications of circulating lipids and their changed bioactivity.

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.

Activation of skeletal muscle protein breakdown following consumption of soyabean protein in pigs.

Diets with protein of inferior quality may increase protein breakdown in skeletal muscle but the experimental results are inconsistent. To elucidate the relationship, pigs were fed isoenergetic and isonitrogenous diets based on soyabean-protein isolate or casein for 15 weeks, with four to six animals per group. A higher plasma level of urea (2.5-fold the casein group value, P = 0.01), higher urinary N excretion (2.1-fold the casein group value, P = 0.01), a postabsorptive rise in the plasma levels of urea, 3-methylhistidine and isoleucine in soyabean protein-fed pigs suggested recruitment of circulatory amino acids by protein breakdown in peripheral tissues. Significant differences between dietary groups were detected in lysosomal and ATP-dependent proteolytic activities in the semimembranosus muscle of food-deprived pigs. A higher concentration of cathepsin B protein was found, corresponding to a rise in the cathepsin B activity, in response to dietary soyabean protein. Muscle ATP-stimulated proteolytical activity was 1.6-fold the casein group value (P = 0.03). A transient rise in the level of cortisol (2.9-times the casein group value, P = 0.02) occurred in the postprandial phase only in the soyabean group. These data suggest that the inferior quality of dietary soyabean protein induces hormonally-mediated upregulation of muscle protein breakdown for recruitment of circulatory amino acids in a postabsorptive state.

Thiazolidinedione-induced activation of the transcription factor peroxisome proliferator-activated receptor gamma in cells adjacent to the murine skeletal muscle: implications for fibroblast functions.

Nuclear peroxisome proliferator-activated receptor gamma (PPARgamma) is the target of antidiabetogenic thiazolidinediones (TZD). However, recent studies failed to show that TZD has an effect in vitro on insulin-regulated glucose uptake in skeletal muscles, the major site of glucose disposal. The potential effects of TZD on cells adjacent to skeletal muscles are not well characterized but may be involved in TZD's actions. Hence, we studied these cells from mice treated with the carrier and with the TZD ciglitazone (9 nmol/g body weight). The cells were typified by lipid enrichment (floating adipocytes and macrophages), by the ectopic expression of cellular fibronectin (fibroblasts), fibronectin and PPARgamma (preadipocytes), PPARgamma and CD11b/Mac-1 (active macrophages) as revealed by flow cytometry and immunoblotting. The glucose transporter 4 proteins (GLUT4) and the uptake of glucose and long-chain fatty acids (LCFA) were determined flow cytometrically using fluorescent derivatives of glucose (NBDG) and LCFA (C16-Bodipy). The expression of tumor necrosis factor alpha (TNFalpha) in CD11b/Mac-1-positive and CD11b/Mac-1-negative cells separated by magnetic immunobeads was analyzed. The results showed that TZD treatment upregulated GLUT4 expression, and increased insulin-regulated NBDG uptake and C16-Bodipy binding and influx, at the same time as increasing the quantity of PPARgamma-expressing fibroblasts; this indicates the development of the preadipocyte phenotype. In contrast, TZD lowered the number of adipocytes (0.6-fold compared to the carrier-treated control) perhaps through an action of TNFalpha from CD11b- and PPARgamma-expressing macrophages. The data suggest that the regulatory effects of TZD on energy homeostasis involve two major targets: the PPARgamma-positive fibroblasts whose adipogenic program is promoted, and CD11b-PPARgamma-expressing macrophages which become cytotoxic and fibrogenic because of the effects of TNFalpha on neighboring adipocytes and fibroblasts, respectively.

Research note: is starch utilization for fat deposition in man different from that in animals?

Utilization of starch for fat deposition in humans was investigated by means of the nitrogen-carbon-balance method in a respiration chamber using the difference principle with a basal and a supplemental period. The basal diet was designed to maintain an adequate energy balance of the volunteer and to meet the maintenance requirements of protein, minerals and vitamins. The dietary fat content was minimized to 2.7% of DM. The digestibility of starch energy was determined to be 97%. The estimated efficiency of ME utilization of starch for energy deposition in humans amounted to 75.8%, which was in accordance with former results in pigs and rats. This suggests that in case of an intake of diets rich in carbohydrates and low in fat the utilization of carbohydrates for lipogenesis in humans is similar to that in monogastric mammal animals.

Influence of inhibitors on increase in intracellular free calcium and proliferation induced by platelet-activating factor in bovine oviductal cells.

Oviductal endosalpingeal cells were isolated mechanically from heifers and cultured until there was 100% confluency. The cells were loaded with the Ca(2+)-sensitive fluorochrome, fura-2/acetoxymethylester, and cytosolic free calcium ([Ca2+]i) was monitored by spectrofluorimetry. Platelet-activating factor, at a concentration of 30 nmol l-1, induced an intracellular Ca2+ increase in cultured bovine oviductal cells, mainly via influx from the extracellular space. In fura-2-loaded oviductal cells, different Ca2+ channel blockers were investigated to characterize the pathways responsible for the Ca2+ influx. The negative effects of Ni(2+)-, La(3+)-activated K+ channel blockers, such as apamin and charybdotoxin, and Ca2+ channel blockers, such as dotarizine, on the platelet-activating factor-induced [Ca2+]i increase indicate the minor participation of the voltage-gated Ca2+ channels. TMB-8 and flufenamic acid blocked the platelet-activating factor-induced Ca2+ increase directly on non-selective cationic channels or acted via a Ca2+ release-triggered Ca2+ influx. Platelet-activating factor, at concentrations of 1.25 mumol l-1 and 2.5 mumol l-1, significantly stimulated the proliferation and depolarization of oviductal cells, but 10 mumol l-1 significantly decreased both parameters and exerted a cytotoxic effect on cells. After incubation with TMB-8 or flufenamic acid, the cell proliferation was inhibited in a concentration-dependent manner, with IC50 values of 26.57 mumol l-1 and 95.29 mumol l-1, respectively. The depolarization was significantly inhibited at 50 mumol l-1 for both TMB-8 and flufenamic acid. The results of the present study may contribute to further understanding of the mechanism behind the actions of platelet-activating factor on oviductal cells.

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.

Cellular aspects of the regulation of metabolic rate.

Beyond the energy requirement of maintenance, the assimilated energy, occurring in bioproducts, is linearly proportional to the intake of metabolizable energy in non-underfed conditions. In contrast, resting metabolic rate is differing between individuals within a population of an animal species. As adaptability to changed environmental conditions may play a role, young bulls were exposed to thermoneutral (18 degrees C) and low (4 degrees C) ambient temperatures and were fed at two feeding levels (1.0 and 1.6 times energy requirement in maintenance) to produce metabolic rate differences, using the same animals, metabolic rate was altered by reducing the sympathetic outflow in each case. Expression of sulfonylurea receptors in circulating mononuclear leukocytes and cells from skeletal muscle (m. semitendinosus) was studied by flow cytometry. Changes of metabolic rate at rest corresponded to the portion of cells with sulfonylurea receptors expression. The data from reducing the sympathetic outflow and those from sulfonylurea receptors expression are useful to explain metabolic rate differences among individuals of an animal population.

Expression of sulphonylurea receptors in bovine monocytes from animals with a different metabolic rate.

The aim of this study was to investigate the relationship between expression of sulphonylurea receptors (SUR) and metabolic rate (MR). SUR on monocytes and cells from muscle tissue were detected using fluorescent glibenclamide and flow cytometry. Transmembrane potential differences were detected by oxonol dye fluorescence measurements. A bovine model was used to induce differences in the MR by exposure to different ambient temperatures (4 degrees C and 18 degrees C), by different feeding levels (1.0- and 1.6-fold the metabolizable energy requirement for maintenance) and by alpha2-adrenergic stimulation. We found that cells from skeletal muscle (m. semimembranosus), immunochemically identified as smooth muscle cells, skeletal muscle fibres and monocytes, responded in comparable fashions to glibenclamide and ATP, i.e. with a depolarization, and to cromakalim with a polarization, suggesting that monocytes are useful indicators of regulatory events occurring in muscle cells. Glibenclamide fluorescence was assumed to represent SUR associated with KATP channels. Significant differences were detected in the percentage of depolarized monocytes in the different variants of the model. A linear correlation between monocytes that bound fluorescent glibenclamide and the MR was evident (with a coefficient of determination of 0.94) and was reproducible following reduction of the MR, by alpha2-adrenergic stimulation, suggesting that expression is involved in the regulation of whole-body energy expenditure.

Validation study of a verbal autopsy method for causes of childhood mortality in Namibia.

Verbal autopsy uses a caretaker interview to determine the cause of death. We conducted a study of the major causes of child death in Namibia to determine the validity of this method. A questionnaire, including signs and symptoms of the diagnoses of interest was administered to the caretaker in 135 deaths of children < 5 years old who were identified from hospital records. The 243 diagnoses included malnutrition (77), diarrhoea (73), pneumonia (36), malaria (33), and measles (24). Sensitivity and specificity of various algorithms of reported signs and symptoms were compared to the medical diagnoses. An algorithm for malnutrition (very thin or swelling) had 73 per cent sensitivity and 76 per cent specificity. An algorithm for cerebral malaria (fever, loss of consciousness or convulsion) had 72 per cent sensitivity and 85 per cent specificity, while for all malaria deaths the same algorithm had low sensitivity (45 per cent) and high specificity (87 per cent). For diarrhoea, loose or liquid stools had high sensitivity (89 per cent), but low specificity (61 per cent). Cough with dyspnoea or tachypnoea had 72 per cent sensitivity and 64 per cent specificity. An algorithm for measles (age > or = 120 days, rash) had 71 per cent sensitivity and 85 per cent specificity. The study results suggest verbal autopsy data can be useful to ascertain the leading causes of death in childhood, but may have limitations for health impact evaluation.

Flow-cytometric analysis of oxidative and proteolytical activities in tissue-associated phagocytes from normal and hypertrophic muscles.

The study was conducted by the known tendencies of increased stress-susceptibility and metabolic disorders in individuals with hypertrophied muscles due to innate factors or intensive exercise which can induce the overtraining syndrome. Using an animal model, muscle-associated cells from normal (N) and hypertrophic (H) skeletal muscle (m.semitendinosus) were examined in their resting and phorbolmyristate acetate (PMA) stimulated oxidation of dihydrorhodamine (DHR) as well as cathepsin B and L activities. Phagocytes were phenotyped by their casein receptors (CR) and fibroblasts by their surface collagens (I and IV). The portion of CR-cells in single cell suspension was 4-8% and 1-3% in H and N. The CR-cells were enriched by 200 g centrifugation and cultured for 5 days with and without cortisol (C), norepinephrine (NE) and indomethacin (I). NE suppressed dose-dependently CR-expression in N, with increase in H occurring. C, NE and I elevated cathepsin activities only in N. PMA stimulated DHR oxidation in H and N 5- and 2-fold. Only the oxidative rate in N reacted to C, NE and I significantly. The data suggest that the response of muscle-associated cells from hypertrophied and normal muscles to signals released in stress-coping significantly differs.

Alterations in the visual cortex receptor pattern by operant conditioning in a reward paradigm.

The aim of the study was to investigate the impact of operant conditioning on the receptor pattern in the visual cortex of calves. A reward paradigm was used to induce conditioned preference for colours. Binding sites in visual cortex specimens from conditioned and naive animals were assayed in vitro on cellular basis after dissociation of the tissue by collagenase, incubation with fluorescent ligands and flow-cytometry for fluorescence analysis. The cellular counts were subdivided according to sedimentation at 200 g as well as via the flow-cytometrical histogram by size and granularity. Binding sites of dopamine D1 and D2 receptor subtypes, glucocorticoids, opioids, casein as well as glycine and N-methyl-D-aspartic acid (NMDA) were detected by fluorescent molecular probes. In displacing naloxone fluorescein from NMDA- and mu-opioid receptors NMDA and meth-enkephalin were used. Comparisons between portions of fluorescent cellular counts from visual cortex tissue of conditioned and naive animals revealed a small increase (1.2-fold, P < 0.05) in opioid receptors of large and high granulated counts, bearing > 80% D1 receptors, and a decrease (0.70-fold, P < 0.05) in less granulated counts with variable portion of D1 receptors. Conditioning resulted in higher and lower displacing rates by meth-enkephalin and NMDA, resp., and in a reduce in counts with dopamine D1 (0.8-fold, P < 0.05), glycine and glucocorticoid binding sites (0.6-fold in both cases, P < 0.01). A tendency of elevated phagocyte marker expression occurred in high granulated counts. The data suggest that conditioning is accompanied with significant and in part marked change in binding sites studied. Induction of scavenger activity may parallel this process. As cellular portion with glycine and glucocorticoid receptors were most markedly altered by conditioning, the neurochemical needs of the used paradigm seem to focus to motility-related functions.

[Results from measurements of nitrogen and energy metabolism in adaptive different cattle]. / Ergebnisse aus Stickstoff- und Energieumsatzmessungen an adaptiv differenten Rindern.

In experiments with Galloway (G), Highland (H) and Black-White Dairy (B) cattle no significant differences between the breeds were measured in the energy and nutrient digestibility and energy metabolizability of rations with high variation in the nutrient composition. In H and B cattle no differences existed in digestibility in relation to the environmental temperature (30, 18 and 3 degrees C). Lowering the environmental temperature from 18 to 12 and 4-6 degrees C resulted in no changes of heat production in G and H but in B heat production increased about 6% and 20% respectively.

Prolactin heterogeneity: a limitation on the evaluation of results from prolactin assays due to differences in immunoassays and the different bioactivities of prolactin forms.

Prolactin exists in biological fluids in several molecular forms. This raises two questions: (1) whether the assay of prolactin by immunotechniques is valid and reliable and (2) whether the different forms have different physiological roles, which might be exploited to improve diagnostic accuracy and data interpretation by the use of appropriate methods. To investigate these questions, prolactin from human amniotic fluid was separated, by concanavalin A-Sepharose affinity chromatography, into bound, retarded and unbound fractions (bound prolactin fraction, retarded prolactin fraction, unbound prolactin fraction), which were characterized by electrophoresis, immunoblotting and glycan detection blot. Virtually no contamination was found in the bound prolactin fraction, and the unbound prolactin fraction and retarded prolactin fraction were 74-83% pure according to densitometry of the electrophoretic and immunoblot patterns. High variability was found among the individual patterns. Glycan detection in the blotted fractions revealed that the bound prolactin fraction bands corresponding to M(r)25,000-29,000 were weakly glycolysated, whereas the bands of M(r)60,000-64,000 were significantly glycan-positive. Immunoreactive bands of unbound prolactin fraction and retarded prolactin fraction also stained positively for glycans. Using two commercial prolactin kits, the bound prolactin fraction forms were virtually undetectable. To demonstrate that the prolactin forms may depend on the hypothalamic state, two behaviourly different breeds of cattle were used as an animal model for studying hypothalamic activities. The number of immunoreactive bands, representing the prolactin forms, and the change of the forms in response to thyroliberin differed strikingly among the groups. The bioactivity of the forms was examined in bovine granulosa, oviductal, endometrial and spleen cells, and in murine splenocytes, the latter being activated by concanavalin A or allogeneically to create in vitro conditions that may have relevance for situations in vivo. The rate of incorporation of [3H]thymidine in murine splenocytes was dose-dependently enhanced only by bound prolactin fraction. The increase was abolished by purified anti-prolactin antiserum. However, the standard prolactin from the kits inhibited the proliferation even in low dose (1.25 microgram/l) and the inhibition was abolished in part by bound prolactin fraction. Thymidine incorporation into the bovine cells was significantly increased by low concentrations (2 micrograms/l) of unbound prolactin fraction and retarded prolactin fraction. Oviduct epithelial cells and splenocytes were stimulated by unbound prolactin fraction but not by retarded prolactin fraction in a dose of 16 micrograms/l. Thymidine incorporation into granulosa cells was inhibited by retarded prolactin fraction (16 micrograms/l) but not by unbound prolactin fraction.(ABSTRACT TRUNCATED AT 400 WORDS)

The glycosylation as source of the variability in prolactin patterns of individual human amniotic fluids.

Whilst it is known that prolactin has size heterogeneity in human amniotic fluid, no studies exist concerning the heterogeneity between individual amniotic fluids and the molecular properties of the prolactin forms which are connected with such a variability. To elecit this, prolactin forms from amniotic fluid taken from 6 full-term deliveries and processed separately, were fractionated by ConA-Sepharose, and characterized by SDS-PAGE, immunoblotting, and glycan detection of blotted prolactins. Recording the absorbance at 280 nm, dimers in the wash of the ConA-Sepharose column and in the elution with methyl-alpha-D-glucoside were evident in 3 fluids. The proteins of the chromatographically more rapid and retarded migrating fractions, generating the first dimer, of unbound material consisted on average of 74-83% and 85-90% of prolactin forms. The eluted fractions (bound material) contained 10-27% of the prolactin amount of the unbound fractions. The electropherograms using 12.5% gels indicated identical patterns of bands in repeated experiments per fluid but variability between the charges. Thus, the apparent molecular masses (kDa) and the proportions to the total prolactin (%) were 14.5-16 (8-12%), 20-23 (3-5%), 24 (4-8%), 25 (6-10%), 27 (19-25%), 28-29 (6-9%), 39-43 (0.5-1.0%), 60-64 (12-19%), 87-90 (8-11%) and 117-140 (2-7%) in nonreducing electrophoretical conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

[Possible prognostic criteria for the evaluation of the susceptibility to septic diseases increased by stress in swine]. / Mögliche prognostische Kriterien zur Beurteilung der bei Schweinen sich durch Belastung erhöhenden Anfälligkeit gegenüber septischen Erkrankungen.

A test to assess leukocyte function developed in our laboratory and based on effector mediated alterations in the cell volume was applied to study the effect of stress on the immune status of pregnant pigs. Effectors selected were prolactin, opsonized particles (Zymosan) and Zymosan together with the autologous plasma. The pigs were exposed to two stress situation: 1. fixation for an ear vein catheterization and 2. injuries by laparotomy. The investigations demonstrated that--following the stress situation 1--the effector induced alterations in the cell volume gave an indication of the susceptibility to septic inflammatory processes subsequent to the laparotomy (stress situation 2) which was carried out about 1 week after the fixation (stress situation 1). Measuring the humoral immunocompetence CRP, C3c, alpha-2-macroglobulin levels in the blood plasma had no or little prognostic value concerning the susceptibility to or the onset of the disease.