Neurotrophic Factors in the Treatment of Acute Brain Hypoxia Secondary to Cardiac Arrest: a Case Report.

Finding neuroprotective agents to counteract the deleterious effects of hypoxia on neuronal cells successfully is one of the most critical targets of clinical research since preclinical studies have identified potential neuroprotective strategies. In clinical practice, amantadine and piracetam are used with reasonable success. We present the cases of three patients with acute brain hypoxia secondary to cardiac arrest, to whom Cerebrolysin was added to the standard neuroprotective treatment regimen, leading to a notable improvement in functional outcome.

Myokine irisin-induced protection against oxidative stress in vitro. Involvement of heme oxygenase-1 and antioxidazing enzymes superoxide dismutase-2 and glutathione peroxidase.

Irisin is a recently discovered myokine reported as protective protein released from exercising skeletal muscles. Although myokines were recently reported to possess the antioxidizing properties, the impact of irisin on the functions of macrophages with respect to its anti-inflammatory potential has not been fully elucidated. Here, we determined the ability of irisin to interact with reactive oxygen species (ROS) in RAW 264.7 murine macrophages. The macrophages were pre-incubated with irisin (0 - 50 nM), some of which had undergone additional co-incubation with bacterial lipopolysaccharide (LPS) (100 ng/ml). Cell viability, the reactive oxygen species scavenging potential as well as the mRNA and protein expression of key oxidative stress factors such as superoxide dismutase 1 (SOD-1), superoxide dismutase 2 (SOD-2), glutathione peroxidase (GSH-Px), catalase 9 (Cat-9), nuclear factor (erythroid-derived 2)-like 2-related factor (Nrf2) and heme oxygenase-1 (HO-1) were evaluated. We found that irisin applied in a concentration of 50 nM significantly attenuated the production of harmful H2O2 and this effect appears to be mediated by a significant increase in the expression of key enzymes involved with antioxidative stress pathways including SOD, GSH-Px and Cat-9 predominantly observed after stimulation of these cells with LPS. We conclude that 1) irisin exhibits a potent antioxidant and anti-inflammatory activities in non-stimulated and LPS-stimulated isolated murine macrophages in vitro, and 2) this protective and antioxidative activity of irisin in vitro might be considered as an important component of protective action of this peptide in vivo, especially under condition of exercise.

New insight into the direct anti-inflammatory activity of a myokine irisin against proinflammatory activation of adipocytes. Implication for exercise in obesity.

A biological activity of myokine irisin, has been intensively investigated in the context of a browning process occurring in white adipose tissue, but its role as a modulator of immune response has been little studied. The aim of our study was to determine the impact of irisin (0 - 100 nM) on pro-inflammatory activation of adipocyte 3T3 L1 cell line. Irisin reduced in a concentration-dependent manner the expression and activity of major proinflammatory cytokines, e.g. tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) expression and their secretion into cell medium. Moreover, irisin enhanced adiponectin synthesis reversing the effect of the lipopolysaccharide (LPS)-induced attenuation of this adipokine expression. The opposite effect was observed for leptin whose expression increased by LPS and this effect was suppressed by irisin application. A decreased phosphorylation and activation of nuclear factor kappa B (NFκB) in the presence of irisin suggests that mechanism of action irisin involves the inhibition of an inflammatory transcription factor. Irisin exerts also an inhibitory effect on macrophage migration toward chemoattractants present in adipocyte supernatants. Among the specific molecules secreted by adipocytes was monocyte chemotactic protein 1 (MCP-1) whose expression was suppressed by irisn. In majority of experiments irisin was effective in 100 nM concentration but in some of them the inhibitory effects occurred already in a concentration of 50 nM of this peptide. This study for the first time showed that adipocytes are directly affected by irisin and provides an evidence on anti-inflammatory action of irisin on fat cells.

Effects of time of day and the wingate test on appetite perceptions, food intake and plasma levels of adipokines.

It has been demonstrated that several aspects of adipose-related physiology including adipokine release, exhibit daily oscillations. Physical exercise exerts a strong influence on adipokine release and a possible reverse disruption of peripheral circadian clocks. The aim of this study was to establish the effects of time of day and the Wingate test on appetite perception, food intake and plasma levels of adipokines. Twenty-four moderately active non-smoking males (mean ± S.D. age: 27.1 ± 3.1 years; height: 1.79 ± 0.1 m; weight: 76.1 ± 11.7 kg) were recruited for this study and divided in two groups; one fed with an ad libitum test meal and another one without an ad libitum test meal. Each subject participated in the following studies performed at 11:00 and 23:00 hours on separate days: 1) Exercise study (ES): a 30-second Wingate Anaerobic Test (WAnT), and 2) sedentary study (SS). Subjects rated their appetite perceptions (hunger and prospective food consumption) on a 100-milimeter visual analogue scale (VAS) at baseline, after exercise, after test meal and during the postprandial/control period. At those time points blood samples were obtained for the measurement of plasma leptin, visfatin and apelin concentrations. Appetite perception and energy intake results at test meal decreased in response to WAnT in comparison with sedentary subjects. Time of day had no statistically significant effect on energy intake but the appetite perception score after test meal at 24:00 hours was statistically higher than that after test meal at 12:00 hours. No significant differences in the tested plasma adipokine concentrations between the trials existed at baseline, however, all plasma adipokine levels at 24:00 hours were higher than those at 12:00 hours. Plasma apelin concentrations after WAnT were significantly higher than its pre-exercise value at 12:00 hours, unlike those at 24:00 hours. Sedentary experiments showed a modest, yet significant, rise in plasma apelin levels after the test meal at 12:00 hours but not after the one at 24:00 hours. There were no significant changes in plasma leptin concentrations after exercise or test meal but a significant decrease in plasma visfatin concentrations after exercise intervention both at the 12:00 hours test and the 24:00 hours test has been observed. Test meals caused a significant rise in visfatin concentrations in sedentary, but not exercise series, in the daytime and nighttime tests. We conclude that time of day is an important aspect to consider in the relationships between exercise, metabolism and appetite. Further studies are needed to explain the specific mechanisms underlying the effects of acute exercise on postprandial physiology at different times of the day.

The impact of physical activity and nutrition on inflammatory bowel disease: the potential role of cross talk between adipose tissue and skeletal muscle.

Crohn's disease and ulcerative colitis are both chronic inflammatory bowel diseases (IBDs) characterized by a cyclical nature, which alternates between active and quiescent states, ultimately impairing a patients' quality of life. The etiology of IBD is not known but it likely involves a combination of genetic predisposition and environmental risk factors. Physical exercise has been suggested to provide protection against the onset of IBD, but there are inconsistencies in the findings of the published literature. Current research recommends exercise to help counteract some IBD-specific complications and preliminary studies suggest that physical activity may be beneficial in reducing the symptoms of IBD. Obesity is becoming more prevalent in patients diagnosed with IBD and may be associated with higher disease activity. There is evidence that adipokines are involved in the inflammatory and metabolic pathways. Hypertrophy of the mesenteric white adipose tissue has been long recognized as a characteristic feature of Crohn's disease; however its importance is unknown. Recent data suggest that dysregulation of adipokine secretion by white adipose tissue is involved in the pathogenesis of Crohn's disease. Skeletal muscle was shown to produce biologically active myokines, which could be a important contributor to the beneficial effects of exercise. There is mounting evidence for the bi-directional endocrine cross talk between adipose tissue and skeletal muscle. The objective of the present review is to explore the role of exercise and its impact on IBD. Also, we discuss how current discoveries regarding the importance of adipokines and myokines and their cross talk expand our view of the pathological changes and the therapeutic options for IBD.

Overfeeding and underfeeding have detrimental effects on oocyte quality measured by in vitro fertilization and early embryonic development in sheep.

To determine effects of maternal diet on in vitro fertilization (IVF) and early embryonic development, ewes (n = 48) were divided into control, overfed (ad libitum feeding), and underfed (60% of control) nutritional planes for 8 wk before oocyte collection. Follicular development was induced by twice-daily injections of FSH on days 13 and 14 of the estrous cycle, and ovaries and blood samples were collected on day 15 of the estrous cycle. During the 8-wk experiment, for control ewes BW and BCS did not change, but for overfed ewes mean (± SEM) BW and BCS increased (11.8 ± 1.1 kg and 2.0 ± 0.1, respectively) and for underfed ewes decreased (14.2 ± 0.9 kg and 0.7 ± 0.1, respectively). The number of follicles was determined; oocytes were collected and subjected to in vitro maturation and fertilization. After IVF, developing embryos were evaluated throughout the 8-d culture period. The proportion of cleaved oocytes after IVF and developing morula and blastocyst were less (P < 0.0001) in overfed and underfed ewes than in control ewes. However, number of visible follicles, total number of oocytes, number of healthy oocytes, and percentage of healthy oocytes were similar for control, overfed, and underfed ewes. Serum insulin concentration was greater (P < 0.05) in overfed ewes than in underfed ewes, estradiol 17-ß (E(2)) concentration was greater (P < 0.05) in underfed ewes than in overfed ewes, but triiodothyronine (T(3)) and thyroxine (T(4)) concentrations were similar in all treatment groups. These data show that inadequate feeding has a negative effect on oocyte quality which results in lower oocyte cleavage after IVF and morula and blastocyst formation; overfeeding increased serum insulin and underfeeding increased serum E(2) but not T(3) or T(4). These data emphasize the importance of diet for reproductive and metabolic functions. Furthermore, the mechanisms through which enhanced or decreased energy in diet affect oocyte quality and serum insulin and E(2) concentrations remain to be elucidated.

Agronomic biofortification of cereal crop plants with Fe, Zn, and Se, by the utilization of coal fly ash as plant growth media.

The implementation of agronomic biofortification of cereal crops with Fe, Zn, and Se appears to be a rapid and simple solution to the deficiency of these elements in soils and plants. These deficiencies are a reason for serious public health concerns. Low levels of Fe, Zn, and Se are important soil constraints to crop production, especially in the developing world. In our study we planted six cereal crops on soil control and different coal combustion residues, naturally rich in these micronutrients. Plants were harvested and chemically analyzed for Fe, Zn, and Se concentration using ICP. Six plant species have been tested including barley (Hordeum vulgare), Jerry oats (Avena sativa), rye (Secale cereale), wheat (Triticum aestivum), perennial ryegrass (Lolium multiflorum), and ReGreen (wheat x wheatgrass hybrid (Triticum aestivum x Thinopyrum intermedium). All tested plants were able to establish growth on coal ash based growth media, and accumulated significant amounts of Fe, Zn, and Se. It supported our hypothesis, that phytoremediation of coal ash piles may serve also as agronomic biofortification of plants, especially cereal crops cultivated on coal fly ash (FA).

Expression of gap junctional connexin proteins in ovine fetal ovaries: effects of maternal diet.

Gap junctions have been implicated in the regulation of cellular metabolism and the coordination of cellular functions during growth and differentiation of organs and tissues, and gap junctions play a major role in direct cell-cell communication. Gap junctional channels and connexin (Cx) proteins have been detected in adult ovaries in several species. Furthermore, it has been shown that several environmental factors, including maternal diet, may affect fetal organ growth and function. To determine whether maternal diet affects expression of Cx26, Cx32, Cx37, and Cx43 in fetal ovaries, sheep were fed a maintenance (M) diet with adequate (A) selenium (Se) or high (H) Se levels from 21 d before breeding to day 132 of pregnancy. From day 50 to 132 of pregnancy (tissue collection day), a portion of the ewes from the ASe and HSe groups was fed a restricted (R; 60% of M) diet. Sections of fetal ovaries were immunostained for the presence of Cxs followed by image analysis. All four Cxs were detected, but the distribution pattern differed. Cx26 was immunolocalized in the oocytes from primordial, primary, secondary, and antral follicles; in granulosa and theca layers of secondary and antral follicles; stroma; and blood vessels. Cx32 was in oocytes, granulosa, and theca cells in a portion of antral follicles; Cx37 was on the borders between oocyte and granulosa/cumulus cells of primordial to antral follicles and in endothelium; and Cx43 was on cellular borders in granulosa and theca layers and between oocyte and granulosa/cumulus cells of primordial to antral follicles. Maternal diet affected Cx26 and Cx43 expression, Cx26 in granulosa layer of antral follicles was decreased (P < 0.01) by HSe in the M and R diets, and Cx43 in granulosa layer of primary and granulosa and theca of antral follicles was increased (P < 0.05) by the M diet with HSe. Thus, Cxs may be differentially involved in regulation of fetal ovarian function in sheep. These data emphasize the importance of maternal diet in fetal growth and development.

Antioxidant capacity of 3D human skin EpiDerm model: effects of skin moisturizers.

The objective of this study was to determine the effects of skin moisturizers on total antioxidant capacity (TAC) of human skin using EpiDerm model. Three different skin moisturizers containing antioxidant ingredients (samples 1-3) or aloe vera extract were topically applied to EpiDerm units and incubated for 2 and 24 h to determine acute and longer-term effects of applied samples on TAC and glutathione peroxidase activity in medium and/or homogenized skin tissues. Total antioxidant capacity in medium and skin homogenates was enhanced (P < 0.0001) by gel containing antioxidant ingredients (sample 2) after 2 and 24 h of incubation. Total antioxidant capacity in medium was also enhanced (P < 0.001) by cream containing antioxidant ingredients (sample 3) after 24 h of incubation. Overall, TAC in medium was greater (P < 0.02) after 24 h than 2 h of incubation. Skin moisturizer cream with high antioxidant levels determined by using oxygen radical absorbance capacity testing (sample 1) and aloe vera extract did not affect TAC. Glutathione peroxidase activity was enhanced (P < 0.0001) in medium and skin homogenates by sample 2 but not by any other sample. These data demonstrate high potential of gel and cream (samples 2 and 3) containing antioxidant ingredients in enhancing antioxidant capacity of EpiDerm which will likely contribute to overall skin health. Results of this experiment will help to better understand mechanisms of effects of skin moisturizers containing antioxidant ingredients on skin function at the tissue level and to establish effective strategies for skin protection and clinical treatments of skin disorders and possibly healing wounds.

Effects of plane of nutrition on in vitro fertilization and early embryonic development in sheep.

Nutrition has been shown to influence several reproductive functions, including hormone production, oocyte competence and fertilization, and early embryonic development. To determine the effects of maternal diet on in vitro fertilization (IVF) and early embryonic development, ewes (n = 18; 47.0 +/- 1.5 kg of initial BW) were divided into control and underfed (60% of control) nutritional planes for 8 wk before oocyte collection. Pelleted diets containing 2.4 Mcal of ME/kg and 13% CP (DM basis) were fed once daily. During the first 4-wk acclimation phase, control and underfed ewes were fed 1,000 and 600 g/d, respectively. From wk 4 to 8, control (adequate) ewes were fed to maintain BW and offered 720 g/d, whereas underfed ewes received 432 g/d (60% restricted). Synchronization of estrus was performed using progestagen sponges for 14 d. Follicular development was induced by twice daily injections of FSH on d 13 (5 units/injection) and 14 (4 units/injection) of the estrous cycle. Oocytes were collected from all visible follicles on d 15 of the estrous cycle. After IVF, the proportion of developing embryos was evaluated throughout an 8-d culture period. Under-nutrition decreased (P < 0.006) the rate of cleavage, number of blastocysts per ewe, and rate of blastocyst formation (from 79 to 64%; from 3.3 to 0.8; and from 31 to 8%, respectively). However, the number of visible follicles, total number of oocytes, number of healthy oocytes, percentage of healthy oocytes, number of cleaved oocytes, and morula formation per ewe were similar for control and underfed ewes. These data indicate that undernutrition of donor ewes, resulting in lower BW and BCS, has a negative effect on oocyte quality, which results in lower rates of cleavage and blastocyst formation.

Effects of epidermal growth factor on early embryonic development after in vitro fertilization of oocytes collected from ewes treated with follicle stimulating hormone.

Epidermal growth factor (EGF) has been shown to enhance the in vitro rate of blastocyst formation in several species. Follicular development was induced in ewes (n=15) by twice daily administration of FSH-P on Days 13 and 14 of the estrous cycle. Cumulus oocyte complexes (COCs) were collected from all visible follicles (n=25+/-2.4/ewe) on Day 15. COCs from each ewe were cultured separately for 24h in maturation medium (containing 10% serum, LH, FSH and estradiol) with (8.2+/-0.9 per ewe) or without (7.8+/-0.8 per ewe) EGF (10 ng/ml). Oocytes were then denuded by hyaluronidase treatment, and healthy oocytes were cultured in the presence of frozen-thawed semen in synthetic oviductal fluid (SOF) medium containing 2% sheep serum. After 18-20 h, zygotes were transferred to SOF medium without glucose and cultured for about 36 h until they reached the 4-8 cell stage. Embryos were transferred to SOF medium with glucose for further development. Medium was changed every other day until blastocyst formation on Day 8 of culture (Day 1=day of fertilization). The rate of embryonic development was evaluated throughout the culture period. After maturation, cumulus cells were more expanded in the presence than in the absence of EGF. The rates of fertilization (overall 75.7+/-3.9%) and morula formation (overall 40.6+/-7.1%) were similar (P>0.05) for COCs cultured with or without EGF. However, EGF increased (P<0.01) the number of blastocysts (1.4+/-0.1 versus 0.6+/-0.2 per ewe) and tended to increase (P<0.1) the rate of blastocyst formation (21.0+/-6.6% versus 13.4+/-4.3% per ewe). These data demonstrate that EGF increases blastocyst formation in FSH-treated ewes. Therefore, EGF is recommended as a supplement to maturation medium to enhance embryonic development in vitro in FSH-treated sheep.

Role of leptin in the control of postprandial pancreatic enzyme secretion.

Leptin released by adipocytes has been implicated in the control of food intake but recent detection of specific leptin receptors in the pancreas suggests that this peptide may also play some role in the modulation of pancreatic function. This study was undertaken to examine the effect of exogenous leptin on pancreatic enzyme secretion in vitro using isolated pancreatic acini, or in vivo in conscious rats with chronic pancreatic fistulae. Leptin plasma level was measured by radioimmunoassay following leptin administration to the animals. Intraperitoneal (i.p.) administration of leptin (0.1, 1, 5, 10, 20 or 50 microg/kg), failed to affect significantly basal secretion of pancreatic protein, but markedly reduced that stimulated by feeding. The strongest inhibition has been observed at dose of 10 microg/kg of leptin. Under basal conditions plasma leptin level averaged about 0.15 +/- 0.04 ng/ml and was increased by feeding up to 1.8 +/- 0.4 ng/ml. Administration of leptin dose-dependently augmented this plasma leptin level, reaching about 0.65 +/- 0.04 ng/ml at dose of 10 microg/kg of leptin. This dose of leptin completely abolished increase of pancreatic protein output produced by ordinary feeding, sham feeding or by diversion of pancreatic juice to the exterior. Leptin (10(-10)-10(-7) M) also dose-dependently attenuated caerulein-induced amylase release from isolated pancreatic acini, whereas basal enzyme secretion was unaffected. We conclude that leptin could take a part in the inhibition of postprandial pancreatic secretion and this effect could be related, at least in part, to the direct action of this peptide on pancreatic acini.

Effects of basic fibroblast growth factor (FGF-2) on proliferation of human skin fibroblasts in type II diabetes mellitus.

Skin fibroblasts from patients with diabetes mellitus display abnormalities in cell proliferation. The use of exogenous growth factors on diabetic wounds has been found to stimulate fibroblast proliferation and facilitate wound healing. However, the results of application of FGF-2 alone to diabetic wounds in clinical trials have been disappointing. The objective of this experiment was to study the effects of FGF-2 and media supplements on in vitro proliferation of skin fibroblasts from patients with type II diabetes and nondiabetic controls, and to evaluate the association between fibroblast proliferation and cAMP production. Fibroblast cell lines (n = 5 from diabetic and n = 5 from control individuals) were cultured in DMEM + 20% FBS for 7 days. Cells were then counted, plated into 24-well plates at a concentration of 2 x 10(4) cells/well and incubated for 24 h in DMEM with serum. The next day, medium was changed to serum-free DMEM alone or DMEM with supplements (albumin, transferrin, insulin and hydrocortisone). Cells were cultured in the presence or absence of varying doses of FGF-2 (0, 0.3, 1, 3, 10 and 30 ng/ml) for 72 hrs then counted and medium was collected for cAMP radioimmunoassay. The doubling time for cell number tended to be greater (p < 0.2) for diabetic fibroblasts than for control fibroblasts. The addition of supplements to the medium reduced (p < 0.05) the doubling time for both fibroblast types. FGF-2 stimulated (p < 0.05) proliferation of diabetic fibroblasts only in medium containing supplements. In contrast, FGF-2 stimulated proliferation of control fibroblasts in medium with or without supplements. The maximal effects of FGF-2 on fibroblast proliferation were greater (p < 0.02) in medium with supplements than in medium without supplements. The K(D) of FGF-2 for fibroblast proliferation was greater (p < 0.06) for diabetic than for control fibroblasts, and lower (p < 0.02) for medium with supplements than for medium without supplements. Fibroblasts from patients with diabetes mellitus produced more (p < 0.05) cAMP than control fibroblasts. These results demonstrate that FGF-2 requires the presence of supplements to enhance proliferation of fibroblasts from patients with type II diabetes mellitus. In addition, fibroblasts from diabetic patients showed a greater K(D) for FGF-2 in terms of cell proliferation. These data suggest a defective FGF receptor or down-regulation of the FGF receptor-mediated cascade that leads to cell proliferation. Identifying methods of reducing the K(D) of FGF-2 in stimulating the proliferation of diabetic fibroblasts may improve the clinical response of diabetic wounds to FGF-2.

Effects of second messengers on gap junctional intercellular communication of ovine luteal cells throughout the estrous cycle.

Corpora lutea (CL) from Days 5, 10, and 15 after superovulation were enzymatically dispersed, and a portion of the cells were elutriated to obtain fractions enriched with small or large luteal cells. Mixed, small, and large luteal cell fractions were incubated with no treatment or with agonists or antagonists of cAMP (dbcAMP or Rp-cAMPS), protein kinase C (PKC; TPA or H-7), or calcium (A23187, EGTA, or A23187 + EGTA). The rate of contact-dependent gap junctional intercellular communication (GJIC) was evaluated by laser cytometry. Media were collected for progesterone (P(4)) radioimmunoassay, and luteal cells cultured with no treatment were fixed for immunocytochemistry or frozen for Western blot analysis. Luteal cells from each stage of the estrous cycle exhibited GJIC. The dbcAMP increased (P < 0.05) GJIC for all cell types across the estrous cycle. The Rp-cAMPS decreased (P < 0.05) GJIC for small luteal cells on Day 5 and for all cell types on Days 10 and 15. The TPA inhibited (P < 0.01), but H-7 did not affect, GJIC for all cell types across the estrous cycle. The A23187 decreased (P < 0.05) GJIC for large luteal cells touching only small or only large luteal cells, whereas A23187 + EGTA decreased (P < 0.05) GJIC for all cell types across the estrous cycle. For the mixed and large luteal cell fractions, dbcAMP increased (P < 0.05), but TPA and A23187 + EGTA decreased (P < 0.05), P(4) secretion. The A23187 alone decreased (P < 0.05) P(4) secretion by large, but not by mixed, luteal cells. For all days and cell types, the rate of GJIC and P(4) secretion were correlated (r = 0.113-0.249; P < 0.01). Connexin 43 was detected in cultured luteal cells by immunofluorescence and Western immunoblotting. Thus, intracellular regulators like cAMP, PKC, or calcium appear to regulate GJIC, which probably is an important mechanism for coordinating function of the ovine CL.

Effects of follicle stimulating hormone (FSH) on follicular development, oocyte retrieval, and in vitro fertilization (IVF) in ewes during breeding season and seasonal anestrus.

Administration of FSH increases the number of developing follicles, and affects oocyte health and cleavage rate. To determine the optimal level of FSH treatment, studies were conducted during the normal breeding season and seasonal anestrus. In Experiment 1, ewes were implanted with SyncroMate-B (SMB; norgestomet) for 14 days during the breeding season. Beginning on day 12 or 13 after SMB implantation, ewes were treated with saline (control; n=10), or treated with FSH for two days (2D; n=9) or three days (3D; n=10). In Experiment 2, conducted during seasonal anestrus, ewes were implanted with SMB for 14 days (n=23) or were not implanted (n=26). The SMB-implanted and nonimplanted ewes were assigned to one of three treatments as in Experiment 1: control (n=13), 2D (n=21) or 3D (n=15). In Experiments 1 and 2, ewes were laparotomized to count the number of follicles < or = 3 mm and > 3 mm and to retrieve oocytes. Healthy oocytes from each treatment were used for IVF. In Experiment 3, ewes (n=6) were implanted twice with SMB for 14 days during seasonal anestrus. Ewes were injected with FSH for 2 days, and the oocytes were collected and fertilized as in Experiments 1 and 2. In Experiment 1, FSH-treatment increased (P < 0.05) the number of follicles > 3 mm, the number of oocytes retrieved from follicles < or = 3 mm and > 3 mm, the proportion of healthy oocytes, and the number of oocytes used for IVF. Oocytes from control and 2D ewes had greater (P < 0.01) cleavage rates than 3D ewes (68% and 71% vs. 42%). In Experiment 2, implanted and nonimplanted ewes had similar (P > 0.05) numbers of follicles, total oocytes, and healthy oocytes; therefore, data were combined. The FSH treatment increased (P < 0.01) the number of follicles > 3 mm, and the number of oocytes recovered from follicles > 3 mm. The recovery rate of oocytes and the percentage of healthy oocytes were similar for control and FSH-treated ewes. The cleavage rate in Experiment 2 ranged from 4 to 16%. In Experiment 3, the cleavage rate for ewes treated twice with SMB was 27% which tended to be greater (P < 0.07) than for the 2D ewes that received one SMB implant in Experiment 2. These data indicate that FSH increased the number of developing follicles and the number of healthy oocytes retrieved from ewes during the breeding season and seasonal anestrus. However, cleavage rates during seasonal anestrus were lower than during the normal breeding season in both FSH-treated and control ewes. Treatment of ewes for 2 days with FSH resulted in a greater cleavage rate than treatment of ewes for 3 days.

The effects of ammonia on pancreatic enzyme secretion in vivo and in vitro.

BACKGROUND: Recent studies clearly demonstrate that Helicobacter pylori (H. pylori) infection of the stomach causes persistent elevation of ammonia (NH3) in gastric juice leading to hypergastrinemia and enhanced pancreatic enzyme secretion. METHODS: The aim of this study is to evaluate the influence of NH4OH on plasma gastrin level and exocrine pancreatic secretion in vivo in conscious dogs equipped with chronic pancreatic fistulas and on secretory activity of in vitro isolated acini obtained from the rat pancreas by collagenase digestion. The effects of NH4OH on amylase release from pancreatic acini were compared with those produced by simple alkalization of these acini with NaOH. RESULTS: NH4OH given intraduodenally (i.d.) in increasing concentrations (0.5, 1.0, 2.0, 4.0, or 8.0 mM/L) resulted in an increase of pancreatic protein output, reaching respectively 9%, 10%, 19%, 16% and 17% of caerulein maximum in these animals and in a marked increase in plasma gastrin level. NH4OH (8 x 0 mM/L, i.d.) given during intravenous (i.v.) infusion of secretin (50 pmol/kg-h) and cholecystokinin (50 pmol/kg-h) reduced the HCO3 and protein outputs by 35% and 37% respectively, as compared to control obtained with infusion of secretin plus cholecystokinin alone. When pancreatic secretion was stimulated by ordinary feeding the same amount of NH4OH administered i.d. decreased the HCO3- and protein responses by 78% and 47% respectively, and had no significant effect on postprandial plasma gastrin. In isolated pancreatic acini, increasing concentrations of NH4OH (10(-7)-10(-4) M) produced a concentration-dependent stimulation of amylase release, reaching about 43% of caerulein-induced maximum. When various concentrations of NH4OH were added to submaximal concentration of caerulein (10(-12) M) or urecholine (10(-5) M), the enzyme secretion was reduced at a dose 10(-5) M of NH4OH by 38% or 40%, respectively. Simple alkalization with NaOH of the incubation medium up to pH 8.5 markedly stimulated basal amylase secretion from isolated pancreatic acini, whereas the secretory response of these acini to pancreatic secretagogues was significantly diminished by about 30%. LDH release into the incubation medium was not significantly changed in all tests indicating that NH4OH did not produce any apparent damage of pancreatic acini and this was confirmed by histological examination of these acini. CONCLUSIONS: 1. NH4OH affects basal and stimulated pancreatic secretion. 2. The excessive release of gastrin may be responsible for the stimulation of basal pancreatic enzyme secretion in conscious animals, and 3. The inhibitory effects of NH4OH on stimulated secretion might be mediated, at least in part, by its direct action on the isolated pancreatic acini possibly due to the alkalization of these acini.

Cell-to-cell communication and expression of gap junctional proteins in human diabetic and nondiabetic skin fibroblasts: effects of basic fibroblast growth factor.

Wound healing involves the interactions of many cell types, and is controlled in part by growth factors. Intercellular communication mediated by gap junctions is considered to play an important role in the coordination of cellular metabolism duringthe growth and development of tissues and organs. Basic fibroblast growth factor (bFGF), known to be important in wound healing, has been found to increase Cx43 expression and intercellular communication in endothelial cells and cardiac fibroblasts. It has been proposed that an increased coupling is necessary for the coordination of these cells in wound healing and angiogenesis, and that one of the actions of bFGF is to modulate intercellular communication. The aim of our study was to evaluate the effects of bFGF on gap junctional intercellular communication (GJIC) in vitro, and the presence of gap junctional proteins connexin (Cx) 26, Cx32, and Cx43 in fibroblasts of diabetic and nondiabetic individuals. Fibroblast cell lines (n = 10) were cultured for 3 d in serum-free media with or without bFGF (3 ng/mL). Cells were evaluated for the rate of GJIC by using laser cytometry, and for the presence of Cx26, Cx32, and Cx43 by immunohistochemical and Western analyses. All cell types communicated via contact-dependent mechanisms. The rate of GJIC was greater (p < 0.01) for diabetic than for nondiabetic fibroblasts (4.1 +/- 0.01 vs 3.3 +/- 0.01%/min). bFGF increased (p < 0.01) the rate of GJIC for diabetic (4.9 +/- 0.01 vs 4.1 +/- 0.01%) and nondiabetic (4.1 +/- 0.01 vs 3.3 +/- 0.01%) fibroblasts. Immunohistochemistry identified Cx26 in the cytoplasm, Cx32 was not detected, and Cx43 was present on the cellular borders in all cultures. Image analysis of immunofluorescent staining demonstrated that bFGF increased (p < 0.05) Cx43 expression in diabetic and nondiabetic fibroblasts. Western immunoblot analysis revealed bands at 43-46 kD that were similar in volume for diabetic and nondiabetic fibroblasts. Thus, gap junctions involving Cx43 and GJIC among fibroblasts appear to be targets for bFGF. Fibroblasts of diabetic individuals appear to have an increased rate of cell-cell coupling, correlating with a decreased rate of proliferation.

Gap junctional proteins, connexin 26, 32, and 43 in sheep ovaries throughout the estrous cycle.

Ovarian follicles from days 13, 14, 15, and 16 and corpora lutea (CL) from days 2, 4, 8, 12, and 15 of the estrous cycle were evaluated for the presence of connexins by immunohistochemistry. In addition, CL from days 5, 10, and 15 of the estrous cycle were used for immunofluorescent detection of Cx43 followed by image analysis, and for Western immunoblot. In all tissues, staining for all connexins appeared punctate, indicating the presence of assembled gap junctions. Cx26 was present in the ovarian surface epithelium, stroma, and blood vessels within the stroma and hilus, and in the CL. In healthy antral follicles, Cx26 was present only in the theca layer, whereas Cx43 was present in granulosa and theca layers. In the majority of atretic follicles, connexins were not detected, but in 13% of the atretic follicles, Cx43 was present in the theca layer. Cx32 was detected in the blood vessels of ovarian stroma and in the CL, and Cx43 was detected in the CL. Localization and/or expression of connexins depended on stage of luteal development. Western analysis demonstrated that expression of Cx32 in luteal tissues was similar across the estrous cycle. The area of positive staining for Cx43 and expression of Cx43 in luteal tissues decreased (p < 0.05) as the estrous cycle progressed. The pattern of expression of connexins indicates that gap junctional proteins may be important in the regulation of folliculogenesis and follicular atresia, as well as growth, differentiation, and regression of the CL.

Gap junctional intercellular communication of bovine luteal cells from several stages of the estrous cycle: effects of prostaglandin F2 alpha, protein kinase C and calcium.

Cellular interactions mediated by both contact-dependent and contact-independent mechanisms are probably important to maintain luteal function. The present studies were performed to evaluate the effects of luteotropic and luteolytic hormones, and also intracellular regulators, on contact-dependent gap junctional intercellular communication (GJIC) of bovine luteal cells from several stages of luteal development. Bovine corpora lutea (CL) from the early, mid and late luteal phases of the estrous cycle were dispersed with collagenase and incubated with no treatment, LH, PGF or LH + PGF (Experiment 1), or with no treatment, or agonists or antagonists of protein kinase C (TPA or H-7) or calcium (A23187 or EGTA; Experiment 2). After incubation, media were collected for determination of progester-one concentrations. Then the rate of GJIC was evaluated for small luteal cells in contact with small luteal cells, and large luteal cells in contact with small luteal cells by using the fluorescence recovery after photobleaching technique and laser cytometry. Luteal cells from each stage of the estrous cycle exhibited GJIC, but the rate of GJIC was least (P < 0.05) for luteal cells from the late luteal phase. LH increased (P < 0.05) GJIC between small luteal cells from the mid and late but not the early luteal phase. PGF increased (P < 0.05) GJIC between small luteal cells from the mid luteal phase and diminished (P < 0.05) LH-stimulatory effects on GJIC between small luteal cells from the late luteal phase. Throughout the estrous cycle, TPA decreased (P < 0.05) the rate of GJIC between large and small, and between small luteal cells, and A23187 decreased (P < 0.05) the rate of GJIC between large and small luteal cells. LH and LH + PGF, but not PGF alone increased (P < 0.05) progesterone secretion by luteal cells from the mid and late luteal phases. Agonists or antagonists of PKC or calcium did not affect progesterone secretion by luteal cells. These data demonstrate that both luteal cell types communicate with small luteal cells, and the rate of communication depends on the stage of luteal development. LH and PGF affect GJIC between small luteal cells during the fully differentiated (mid-luteal) and regressing (late luteal) stages of the estrous cycle. In contrast, at all stages of luteal development, activation of PKC decreases GJIC between small and between large and small luteal cells, whereas calcium ionophore decreases GJIC only between large and small luteal cells. Luteotropic and luteolytic hormones, and intracellular regulators, may be involved in regulation of cellular interactions within bovine CL which likely is an important mechanism for coordination of luteal function.

Gap junctional protein connexin 43 in bovine corpora lutea throughout the estrous cycle.

The present study examined the pattern of expression of the gap junctional protein connexin 43 (Cx43) in bovine corpora lutea (CL) during growth, differentiation, and regression. CL from the early (n = 6), mid- (n = 6), and late (n = 6) luteal phases of the estrous cycle were weighed and divided into several portions. One portion of each CL was frozen in liquid nitrogen for evaluation of protein, DNA, progesterone, and presence of Cx43 by Western immunoblot analysis; another portion was frozen in liquid propane for immunofluorescent staining of Cx43. An additional portion of each CL was dispersed, and the luteal cells were cultured for 2 days, fixed, and used for immunofluorescent staining of Cx43. Weights and DNA, protein, and progesterone contents of CL increased (p < 0.05) from the early to mid-luteal phases and then decreased (p < 0.05) from the mid- to late luteal phases. The ratio of protein to DNA was similar in the early and mid-luteal phases and then decreased (p < 0.05) to the late luteal phase. Western immunoblot analysis revealed bands at 43 kDa that differed in volume (evaluated by densitometry); the early luteal phase volume was greater (p < 0.05) than that at the mid-luteal phase, which was greater (p < 0.05) than that at the late luteal phase. Immunofluorescent staining demonstrated that Cx43 was present in luteal tissues and cultured luteal cells throughout the estrous cycle, and the area of positive staining decreased (p < 0.05) as the estrous cycle progressed. Staining for Cx43 was punctate and localized to the cellular borders. Thus, levels of Cx43 in bovine CL are greatest early in the estrous cycle and are least late in the estrous cycle. These data demonstrate that gap junctions may be important for regulation of luteal growth, differentiation, and regression in the cow.