La dynamique chromatinienne induite par le pic de LH dans les cellules de granulosa chez la souris

La régulation transcriptionnelle des gènes est un processus indispensable sans lequel la diversité phénotypique des cellules ainsi que l’adaptation à leur environnement serait inexistant. L’identification des éléments de régulation dans le génome est d’une importance capitale afin de comprendre les mécanismes gouvernant l’expression des gènes spécifiques à un type cellulaire donné. Ainsi, suite au pic de LH, le follicule ovarien entre dans un programme intensif de différentiation cellulaire, orchestré par des modifications majeures du profile transcriptionnel des cellules de granulosa, déclenchant ultimement l’ovulation et la lutéinisation, processus indispensables à la fertilité femelle. L’hypothèse supportée par cette étude stipule qu’une réorganisation de la structure chromatinienne survient aux régions régulatrices d’une panoplie de gènes dans les heures suivant le pic de LH et qu’en isolant et identifiant ces régions, il serait possible de retrouver des éléments essentiels aux processus d’ovulation et de lutéinisation. Ainsi, en utilisant un protocole standard de superovulation chez la souris, les éléments de régulation se modifiant 4h suivant l’administration de hCG ont été isolés et identifiés dans les cellules de granulosa en utilisant la méthode FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) combinée à un séquençage haut débit. Cette étude a démontré que suite au stimulus ovulatoire, les cellules de granulosa subissent une reprogrammation majeure des éléments de régulation, qui est corrélée avec une modification drastique de leurs fonctions biologiques. De plus, cette étude a mis en évidence une association majoritaire des éléments de régulation à des régions intergéniques distales et à des introns, indiquant que ces régions ont une importance capitale dans la régulation transcriptionnelle dans les cellules de granulosa. Cette étude a également permis d’identifier une panoplie de régulateurs transcriptionnels reconnus pour être essentiels à la fonction ovarienne, ainsi que leur sites de liaison dans le génome, démontrant que la méthode FAIRE est une méthode assez puissante pour permettre la prédiction d’événements moléculaires précis ayant un sens physiologique réel.

Acute insult of ammonia leads to calcium-dependent glutamate release from cultured astrocytes, an effect of pH.

Hyperammonemia is a key factor in the pathogenesis of hepatic encephalopathy (HE) as well as other metabolic encephalopathies, such as those associated with inherited disorders of urea cycle enzymes and in Reye's syndrome. Acute HE results in increased brain ammonia (up to 5 mM), astrocytic swelling, and altered glutamatergic function. In the present study, using fluorescence imaging techniques, acute exposure (10 min) of ammonia (NH4+/NH3) to cultured astrocytes resulted in a concentration-dependent, transient increase in [Ca2+]i. This calcium transient was due to release from intracellular calcium stores, since the response was thapsigargin-sensitive and was still observed in calcium-free buffer. Using an enzyme-linked fluorescence assay, glutamate release was measured indirectly via the production of NADH (a naturally fluorescent product when excited with UV light). NH4+/NH3 (5 mM) stimulated a calcium-dependent glutamate release from cultured astrocytes, which was inhibited after preincubation with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester but unaffected after preincubation with glutamate transport inhibitors dihydrokainate and DL-threo-beta-benzyloxyaspartate. NH4+/NH3 (5 mM) also induced a transient intracellular alkaline shift. To investigate whether the effects of NH4+/NH3 were mediated by an increase in pH(i), we applied trimethylamine (TMA+/TMA) as another weak base. TMA+/TMA (5 mM) induced a similar transient increase in both pH(i) and [Ca2+]i (mobilization from intracellular calcium stores) and resulted in calcium-dependent release of glutamate. These results indicate that an acute exposure to ammonia, resulting in cytosolic alkalinization, leads to calcium-dependent glutamate release from astrocytes. A deregulation of glutamate release from astrocytes by ammonia could contribute to glutamate dysfunction consistently observed in acute HE.

Dopamine Receptor Gene Expression In Streptozotocin Induced Diabetic Rats And Its Role In Insulin Secretion

Diabetes Mellitus is a metabolic disorder associated with insulin deficiency, which not.only affects the carbohydrate metabolism but also is associated with various central and peripheral complications. Chronic hyperglycemia during diabetes mellitus is a major initiator of diabetic microvascular complications like retinopathy, neuropathy, The central nervous system (CNS) neurotransmitters play an important role in the regulation of glucose homeostasis. These neurotransmitters mediate rapid intracellular communications not only within the central nervous system but also in the peripheral tissues. They exert their function through receptors present in both neuronal and non neuronal cell surface that trigger second messenger signaling pathways. Dopamine is a neurotransmitter that has been implicated in various central neuronal degenerative disorders like Parkinson's disease and behavioral diseases like Schizophrenia. Dopamine is synthesised from tyrosine, stored in vesicles in axon terminals and released when the neuron is depolarised. Dopamine interacts with specific membrane receptors to produce its effect. Dopamine plays an important role both centrally and peripherally. The recent identification of five dopamine receptor subtypes provides a basis for understanding dopamine's central and peripheral actions . Dopamine receptors are classified into two major groups : DA D1 like and DA D2 like. Dopamine D1 like receptors consists of DA D1 and DA D5 receptors . Dopamine D2 like receptors consists of DA D2, DA D3 and DA D4 receptors. Stimulation of the DA D1 receptor gives rise to increased production of cAMP. Dopamine D2 receptors inhibit cAMP production, but activate the inositol phosphate second messenger system . Impairment of central dopamine neurotransmission causes muscle rigidity, hormonal regulation , thought disorder and cocaine addiction. Peripheral dopamine receptors mediate changes in blood flow, glomerular filtration rate, sodium excretion and catecholamine release. The dopamine D2 receptors increased in the corpus striatum and cerebral cortex but decreased in the hypothalamus and brain stem indicating their involvement in regulating insulin secretion. Dopamine D2 receptor which has a stimulatory effecton insulin secretion decreased in the pancreatic islets during diabetes. Our in vitro studies confirmed the stimulatory role of dopamine D2 receptors in stimulation of glucose induced insulin secretion. A detailed study at the molecular level on the mechanisms involved in the role of dopamine in insulin secretion, its functional modification could lead to therapeutic interventions that will have immense clinical importance.

Muscarinic M1 and M3 Receptors,IP3 and cGMP Functional Regulation in Streptozotocin Induced Diabetic Rats: Insulin and Somatotropin Induced Rejuvenation as a Function of Age

The present study describes that acetylcholine through muscarinic Ml and M3 receptors play an important role in the brain function during diabetes as a function of age. Cholinergic activity as indicated by acetylcholine esterase, a marker for cholinergic function, decreased in the brain regions - the cerebral cortex, brainstem and corpus striatum of old rats compared to young rats. in diabetic condition, it was increased in both young and old rats in cerebral cortex, and corpus striatum while in brainstem it was decreased. The functional changes in the muscarinic receptors were studied in the brain regions and it showed that muscarinic M I receptors of old rats were down regulated in cerebral cortex while in corpus striatum and brainstem it was up regulated. Muscarinic M3 receptors of old rats showed no significant change in cerebral cortex while in corpus striatum and brainstem muscarinic receptors were down regulated. During diabetes, muscarinic M I receptors were down regulated in cerebral cortex and brainstem of young rats while in corpus striatum they were up regulated. In old rats, M I receptors were up regulated in cerebral cortex, corpus striatum and in brainstem they were down regulated. Muscarinic M3 receptors were up regulated in cerebral cortex and brainstem of young rats while in corpus striatum they were down regulated. In old rats, muscarinic M l receptors were up regulated in cerebral cortex, corpus striatum and brainstem. In insulin treated diabetic rats the activity of the receptors were reversed to near control. Pancreatic muscarinic M3 receptor activity increased in the pancreas of both young and old rats during diabetes. In vitro studies using carbachol and antagonists for muscarinic Ml and M3 receptor subtypes confirmed the specific receptor mediated neurotransmitter changes during diabetes. Calcium imaging studies revealed muscarinic M I mediated Ca2 + release from the pancreatic islet cells of young and old rats. Electrophysiological studies using EEG recording in young and old rats showed a brain activity difference during diabetes. Long term low dose STH and INS treated rat brain tissues were used for gene expression of muscarinic Ml, M3, glutamate NMDARl, mGlu-5,alpha2A, beta2, GABAAa1 and GABAB, DAD2 and 5-HT 2C receptors to observe the neurotransmitter receptor functional interrelationship for integrating memory, cognition and rejuvenating brain functions in young and old. Studies on neurotransmitter receptor interaction pathways and gene expression regulation by second messengers like IP3 and cGMP in turn will lead to the development of therapeutic agents to manage diabetes and brain activity.From this study it is suggested that functional improvement of muscarinic Ml, M3, glutamate NMDAR1, mGlu-5, alpha2A, beta2, GABAAa1 and GABAB, DAD2 and 5-HT 2C receptors mediated through IP3 and cGMP will lead to therapeutic applications in the management of diabetes. Also, our results from long term low dose STH and INS treatment showed rejuvenation of the brain function which has clinical significance in maintaining healthy period of life as a function of age.

Glutamatergic Nmda And Ampa Receptors Functional Regulation In Streptozotocin Induced Diabetic Rats: Effect Of Vitamin D3 And Curcumin Supplementation

The present study was designed to investigate the protective effect of curcumin and vitamin D3 in the functional regulation of glutamatergic NMDA and AMPA receptors in streptozotocin (STZ) induced diabetic rats. Alterations in glutamatergic neurotransmission in the brain were evaluated by analyzing the glutamate content, glutamate receptors - NMDA and AMPA receptors binding parameters and gene expression, GAD and GLAST gene expression. Immunohistochemistry studies using confocal microscope were carried out to confirm receptor density and gene expression results of NMDA and AMPA receptors. The role of glutamatergic receptors in pancreas was studied using the following parameters; glutamate content, GLAST expression, glutamate receptors - NMDA and AMPA receptor binding and gene expression. Increasing evidence in both experimental and clinical studies suggests that oxidative stress plays a major role in the pathogenesis of diabetes. In the present study SOD assay and GPx gene expression were done to evaluate the activity of antioxidant enzymes in the brain regions and pancreas. NeuroD1 and Pdx1 gene expression were performed in pancreas of experimental rats to evaluate pancreatic islet survival. Gene expression profiles of caspase 8, Bax, and Akt in brain regions and pancreas were studied to understand the possible mechanism behind curcumin and vitamin D3 mediated neuroprotection and islet survival. Gene expression studies of vitamin D3 receptor localisation in the pancreas was done to understand the mechanism of vitamin D3 in insulin secretion. Curcumin and vitamin D3 mediated insulin secretion via Ca2+ release were studied using confocal microscope.

Serotonin Receptor Gene Expression And Insulin Function In Streptozotocin Induced Diabetic Rats

Recent studies have established a fimctional correlation of serotonergic and adrenergic function in the brain regions with insulin secretion in diabetic rats (Vahabzadeh et al., 1995). Administration of 5-HT”. agonist 8-OH-DPAT to conscious rats caused an increase in blood glucose level. This increase in blood glucose is due to inhibition of insulin secretion by increased circulating EPI (Chaouloff et al., 1990a; Chaouloff et al., 1990d; Chaoulo1T& Jeanrenaud, 1987). The increase in EPI is brought about by increased sympathetic stimulation. This increase can lead to increased sympatho-medullary stimulation thereby inhibiting insulin release (Bauhelal & Mir, 1993, Bauhelal & Mir, 1990a; Chaouloffet al., 1990d). Also, studies have shown that Gi protein in the liver has been decreased in diabetes which will increase gluconeogenesis and glycogenolysis thereby causing hyperglycaemia (Pennington, 1987). Serotonergic control is suggested to exert different effects on insulin secretion according to the activation of different receptor subclasses (Pontiroli et al., 1975). In addition to this mechanism, the secretion of insulin is dependent on the turnover ratio of endogenous 5-hydroxy tryptophan (5-HTP) to 5-HT in the pancreatic islets (Jance er al., 1980). The reports so far stated does not explain the complete mechanism and the subclass of 5-HT receptors whose expression regulate insulin secretion in a diabetic state. Also, there is no report of a direct regulation of insulin secretion by 5-HT from the pancreatic islets even though there are reports stating that the pancreatic islets is a rich source of 5-HT (Bird et al., 1980). Therefore, in the present study the mechanism by which 5-HT and its receptors regulate insulin secretion from pancreatic [3-cells was investigated. Our results led to the following hypotheses by which 5-HT and its receptors regulate the insulin secretion.

Salinity-induced changes in the microbial use of sugarcane filter cake added to soil

Five laboratory incubation experiments were carried out to assess the salinity-induced changes in the microbial use of sugarcane filter cake added to soil. The first laboratory experiment was carried out to prove the hypothesis that the lower content of fungal biomass in a saline soil reduces the decomposition of a complex organic substrate in comparison to a non-saline soil under acidic conditions. Three different rates (0.5, 1.0, and 2.0%) of sugarcane filter cake were added to both soils and incubated for 63 days at 30°C. In the saline control soil without amendment, cumulative CO2 production was 70% greater than in the corresponding non-saline control soil, but the formation of inorganic N did not differ between these two soils. However, nitrification was inhibited in the saline soil. The increase in cumulative CO2 production by adding filter cake was similar in both soils, corresponding to 29% of the filter cake C at all three addition rates. Also the increases in microbial biomass C and biomass N were linearly related to the amount of filter cake added, but this increase was slightly higher for both properties in the saline soil. In contrast to microbial biomass, the absolute increase in ergosterol content in the saline soil was on average only half that in the non-saline soil and it showed also strong temporal changes during the incubation: A strong initial increase after adding the filter cake was followed by a rapid decline. The addition of filter cake led to immobilisation of inorganic N in both soils. This immobilisation was not expected, because the total C-to-total N ratio of the filter cake was below 13 and the organic C-to-organic N ratio in the 0.5 M K2SO4 extract of this material was even lower at 9.2. The immobilisation was considerably higher in the saline soil than in the non-saline soil. The N immobilisation capacity of sugarcane filter cake should be considered when this material is applied to arable sites at high rations. The second incubation experiment was carried out to examine the N immobilizing effect of sugarcane filter cake (C/N ratio of 12.4) and to investigate whether mixing it with compost (C/N ratio of 10.5) has any synergistic effects on C and N mineralization after incorporation into the soil. Approximately 19% of the compost C added and 37% of the filter cake C were evolved as CO2, assuming that the amendments had no effects on the decomposition of soil organic C. However, only 28% of the added filter cake was lost according to the total C and d13C values. Filter cake and compost contained initially significant concentrations of inorganic N, which was nearly completely immobilized between day 7 and 14 of the incubation in most cases. After day 14, N re-mineralization occurred at an average rate of 0.73 µg N g-1 soil d-1 in most amendment treatments, paralleling the N mineralization rate of the non-amended control without significant difference. No significant net N mineralization from the amendment N occurred in any of the amendment treatments in comparison to the control. The addition of compost and filter cake resulted in a linear increase in microbial biomass C with increasing amounts of C added. This increase was not affected by differences in substrate quality, especially the three times larger content of K2SO4 extractable organic C in the sugarcane filter cake. In most amendment treatments, microbial biomass C and biomass N increased until the end of the incubation. No synergistic effects could be observed in the mixture treatments of compost and sugarcane filter cake. The third 42-day incubation experiment was conducted to answer the questions whether the decomposition of sugarcane filter cake also result in immobilization of nitrogen in a saline alkaline soil and whether the mixing of sugarcane filter cake with glucose (adjusted to a C/N ratio of 12.5 with (NH4)2SO4) change its decomposition. The relative percentage CO2 evolved increased from 35% of the added C in the pure 0.5% filter cake treatment to 41% in the 0.5% filter cake +0.25% glucose treatment to 48% in the 0.5% filter cake +0.5% glucose treatment. The three different amendment treatments led to immediate increases in microbial biomass C and biomass N within 6 h that persisted only in the pure filter cake treatment until the end of the incubation. The fungal cell-membrane component ergosterol showed initially an over-proportionate increase in relation to microbial biomass C that fully disappeared at the end of the incubation. The cellulase activity showed a 5-fold increase after filter cake addition, which was not further increased by the additional glucose amendment. The cellulase activity showed an exponential decline to values around 4% of the initial value in all treatments. The amount of inorganic N immobilized from day 0 to day 14 increased with increasing amount of C added in comparison to the control treatment. Since day 14, the immobilized N was re-mineralized at rates between 1.31 and 1.51 µg N g-1 soil d-1 in the amendment treatments and was thus more than doubled in comparison with the control treatment. This means that the re-mineralization rate is independent from the actual size of the microbial residues pool and also independent from the size of the soil microbial biomass. Other unknown soil properties seem to form a soil-specific gate for the release of inorganic N. The fourth incubation experiment was carried out with the objective of assessing the effects of salt additions containing different anions (Cl-, SO42-, HCO3-) on the microbial use of sugarcane filter cake and dhancha leaves amended to inoculated sterile quartz sand. In the subsequent fifth experiment, the objective was to assess the effects of inoculum and temperature on the decomposition of sugar cane filter cake. In the fourth experiment, sugarcane filter cake led to significantly lower respiration rates, lower contents of extractable C and N, and lower contents of microbial biomass C and N than dhancha leaves, but to a higher respiratory quotient RQ and to a higher content of the fungal biomarker ergosterol. The RQ was significantly increased after salt addition, when comparing the average of all salinity treatments with the control. Differences in anion composition had no clear effects on the RQ values. In experiment 2, the rise in temperature from 20 to 40°C increased the CO2 production rate by a factor of 1.6, the O2 consumption rate by a factor of 1.9 and the ergosterol content by 60%. In contrast, the contents of microbial biomass N decreased by 60% and the RQ by 13%. The effects of the inoculation with a saline soil were in most cases negative and did not indicate a better adaptation of these organisms to salinity. The general effects of anion composition on microbial biomass and activity indices were small and inconsistent. Only the fraction of 0.5 M K2SO4 extractable C and N in non-fumigated soil was consistently increased in the 1.2 M NaHCO3 treatment of both experiments. In contrast to the small salinity effects, the quality of the substrate has overwhelming effects on microbial biomass and activity indices, especially on the fungal part of the microbial community.

Causes of legume rotation induced yield increases in cereals grown on West African soils

This study was conducted to investigate soil biological and chemical factors that give rise to cereal yield enhancing effects of legume rotations on sandy, nutrient poor West African soils. The aim was not only to gain more information on the role of legume residues and microorganisms in the soil nutrient cycle. But the study aimed at evaluating if differences in substrate qualities (e.g. root residues) cause changes in the microbial community structure due to specific and highly complex microbe-root-soil interactions. Site and system specific reactions of microorganisms towards rewetting, simulating the onset of rainy season, were observed. Higher respiration rates, higher amounts of microbial biomass carbon (Cmic) and nitrogen (Nmic) as well as higher ergosterol, muramic acid, glucosamine and adenylate concentrations were measured in CL soils of Koukombo and in both soils from Fada. The immediate increase in ATP concentrations after rewetting was likely caused by rehydration of microbial cells where N was not immobilized and, thus, available for plants facilitating their rapid development. Legume root residues led only to slightly better plant performances compared to the control, while the application of cereal roots reduced seedling growth. In contrast to sorghum seedlings, the microbial community did not react to the mineral treatment. Thus the energy supply in form of organic amendments increased microbial indices compared to mineral P application and the control. The results of basal respiration rates, Cmic and Corg levels indicate that the microbial community in the soil from Koukombo is less efficient in substrate use compared to microorganisms in the soil from Fada. However, the continuous carbon input by legume root residues might have contributed to these differences in soil fertility. With the 33P isotopic exchange method a low buffering capacity was detected in both soils irrespective of treatments. Calculated E values (E1min to E1min-1d and E1d-3m) indicated a slowly release of P due to root turnover while applied mineral P is taken up by plants or fixed to the soil. Due to the fact that sorghum growth reacted mainly to the application of mineral P and the microorganisms solely to the organic inputs, the combination of both amendments seems to be the best approach to a sustainable increase of crop production on many nutrient-poor, sandy West African soils. In a pot experiment, were CC and CL soils from Fada and Koukombo were adjusted to the same level of P and N concentrations, crop growth was significantly higher on CL soils, compared to the respective treatments on CC soils. Mycorrhizal infection of roots was increased and the number of nematodes, predominantly free living nematodes, was almost halfed on rotation soils. In conclusion, increased nutrient availability (especially P and N) through the introduction of legumes is not the only reason for the observed yield increasing effects. Soil biological factors seem to also play an important role. In a root chamber experiment the pH gradient along the root-soil-interface was measured at three times using an antimony microelectrode. For Fada soils, pH values were higher on CL than CC soils while the opposite was true for the Koukombo soils. Site-specific differences between Fada and Koukombo soils in N content and microbial community structures might have created varying crop performances leading to the contrasting pH findings. However, the mechanisms involved in this highly complex microbe-root-soil interaction remain unclear.

Apatite-Polymer Composite Particles for Controlled Delivery of BMP-2: In Vitro Release and Cellular Response

Bone morphogenetic protein-2 (BMP-2) has the ability to induce osteoblast differentiation of undifferentiated cells, resulting in the healing of skeletal defects when delivered with a suitable carrier. We have applied a versatile delivery platform comprising a novel composite of two biomaterials with proven track records – apatite and poly(lactic-co-glycolic acid) (PLGA) – to the delivery of BMP-2. Sustained release of this growth factor was tuned with variables that affect polymer degradation and/or apatite dissolution, such as polymer molecular weight, polymer composition, apatite loading, and apatite particle size. The effect of released BMP-2 on C3H10T1/2 murine pluripotent mesenchymal cells was assessed by tracking the expression of osteoblastic makers, alkaline phosphatase (ALP) and osteocalcin. Release media collected over 100 days induced elevated ALP activity in C3H10T1/2 cells. The expression of osteocalcin was also upregulated significantly. These results demonstrated the potential of apatite-PLGA composite particles for releasing protein in bioactive form over extended periods of time.

Bacterial polysaccharides suppress induced innate immunity by calcium chelation

Bacterial pathogens and symbionts must suppress or negate host innate immunity. However, pathogens release conserved oligomeric and polymeric molecules or MAMPs (Microbial Associated Molecular Patterns), which elicit host defenses [1], [2] and [3]. Extracellular polysaccharides (EPSs) are key virulence factors in plant and animal pathogenesis, but their precise function in establishing basic compatibility remains unclear [4], [5], [6] and [7]. Here, we show that EPSs suppress MAMP-induced signaling in plants through their polyanionic nature [4] and consequent ability to chelate divalent calcium ions [8]. In plants, Ca2+ ion influx to the cytosol from the apoplast (where bacteria multiply [4], [5] and [9]) is a prerequisite for activation of myriad defenses by MAMPs [10]. We show that EPSs from diverse plant and animal pathogens and symbionts bind calcium. EPS-defective mutants or pure MAMPs, such as the flagellin peptide flg22, elicit calcium influx, expression of host defense genes, and downstream resistance. Furthermore, EPSs, produced by wild-type strains or purified, suppress induced responses but do not block flg22-receptor binding in Arabidopsis cells. EPS production was confirmed in planta, and the amounts in bacterial biofilms greatly exceed those required for binding of apoplastic calcium. These data reveal a novel, fundamental role for bacterial EPS in disease establishment, encouraging novel control strategies.

Bone morphogenetic proteins (BMP)-4,-6, and-7 potently suppress basal and luteinizing hormone-induced androgen production by bovine theca interna cells in primary culture: Could ovarian hyperandrogenic dysfunction be caused by a defect in thecal BMP signaling?

We reported recently that bovine theca interna cells in primary culture express several type-I and type-II receptors for bone morphogenetic proteins (BMPs). The same cells express at least two potential ligands for these receptors (BMP-4 and - 7), whereas bovine granulosa cells and oocytes express BMP-6. Therefore, BMPs of intrafollicular origin may exert autocrine/paracrine actions to modulate theca cell function. Here we report that BMP-4, - 6, and - 7 potently suppress both basal ( P < 0.0001; respective IC50 values, 0.78, 0.30, and 1.50 ng/ml) and LH-induced ( P < 0.0001; respective IC50 values, 5.00, 0.55, and 4.55 ng/ml) androgen production by bovine theca cells while having only a moderate effect on progesterone production and cell number. Semiquantitative RT-PCR showed that all three BMPs markedly reduced steady-state levels of mRNA for P450c17. Levels of mRNA encoding steroidogenic acute regulatory protein, P450scc, and 3 beta-hydroxysteroid dehydrogenase were also reduced but to a much lesser extent. Immunocytochemistry confirmed a marked reduction in cellular content of P450c17 protein after BMP treatment ( P < 0.001). Exposure to BMPs led to cellular accumulation of phosphorylated Smad1, but not Smad2, confirming that the receptors signal via a Smad1 pathway. The specificity of the BMP response was further explored by coincubating cells with BMPs and several potential BMP antagonists, chordin, gremlin, and follistatin. Gremlin and chordin were found to be effective antagonists of BMP-4 and - 7, respectively, and the observation that both antagonists enhanced ( P < 0.01) androgen production in the absence of exogenous BMP suggests an autocrine/paracrine role for theca-derived BMP- 4 and - 7 in modulating androgen production. Collectively, these data indicate that an intrafollicular BMP signaling pathway contributes to the negative regulation of thecal androgen production and that ovarian hyperandrogenic dysfunction could be a result of a defective autoregulatory pathway involving thecal BMP signaling.

The reproductive response of rams to thyroidectomy: mediation by impaired inhibin feedback rather than a change in LH pulsatility

A series of experiments was conducted to examine the mechanism by which removal of the thyroid glands in seasonally suppressed rams brings about rapid testicular growth. In the first experiment, thyroidectomy at the nadir of the testicular cycle (late winter) initiated testis growth without any detectable change in the extent of spermatogenesis compared with sham-operated controls. The serum concentration of FSH, but not LH, was also markedly increased by thyroidectomy. In the second experiment, serum FSH concentration was again increased by thyroidectomy in late winter but there was no effect of thyroidectomy on LH concentration, LH pulses (measured in frequent blood samples) or testosterone concentration. Furthermore, there was no evidence of a change in central dopaminergic inhibition of GnRH, as measured by the pulsatile LH response to an i.m. injection of the dopaminergic D-2 agonist bromocriptine or antagonist sulpiride. The rapid increase in FSH concentration occurred despite a markedly increased serum inhibin A concentration in thyroidectomized rams. Therefore, the efficacy of inhibin feedback was examined by testing the FSH-suppressive effect of an inhibin preparation (5 ml charcoal-stripped bovine follicular fluid i.v.) in long-term thyroidectomized and thyroid intact castrated rams. Bovine follicular fluid suppressed FSH concentrations in control rams as expected but in marked contrast, was completely without effect in thyroidectomized animals. In castrated rams, the FSH concentration was only marginally increased by thyroidectomy, indicating that there is a major component of the mediation of the effects of thyroidectomy that is testicular in origin. It was concluded that a reduction in the ability of endogenous inhibin to inhibit FSH release at the pituitary, rather than a hypothalamic mechanism, is the primary cause of the stimulation of testis growth by thyroidectomy.

Vacuum-induced bubble formation in liquid-tempered chocolate

Bubble inclusion is one of the fastest growing operations practiced in the food industry. A variety of aerated foods is currently available in supermarkets, and newer products are emerging all the time. This paper aims to combine knowledge on chocolate aeration with studies performed on bubble formation and dispersion characteristics. More specifically, we have investigated bubble formation induced by applying vacuum. Experimental methods to determine gas hold-up (volume fraction of air), bubble section distributions along specific planes, and chocolate rheological properties are presented. This study concludes that decreasing pressures elevate gas hold-up values due to an increase in the number of bubble nuclei being formed and release of a greater volume of dissolved gases. Furthermore, bubbles are observed to be larger at lower pressures for a set amount of gas because the internal pressure needs to be in equilibrium with the surrounding pressures. Temperature-induced changes to the properties of the chocolate have less of an effect on bubble formation. On the other hand, when different fats and emulsifiers are added to a standard chocolate recipe, milk fat was found to increase, significantly, the gas hold-up values and the mean bubble-section diameters. It is hypothesized that this behavior is related to the way milk fats, which contain different fatty acids to cocoa butter, crystallize and influence the setting properties of the final product. It is highlighted that apparent viscosity values at low shear rate, as well as setting behavior, play an important role in terms of bubble formation and entrainment.

G protein ss gamma subunits mediate presynaptic inhibition of transmitter release from rat superior cervical ganglion neurones in culture

The activation of presynaptic G protein-coupled receptors (GPCRs) is widely reported to inhibit transmitter release; however, the lack of accessibility of many presynaptic terminals has limited direct analysis of signalling mediators. We studied GPCR-mediated inhibition of fast cholinergic transmission between superior cervical ganglion neurones (SCGNs) in culture. The adrenoceptor agonist noradrenaline (NA) caused a dose-related reduction in evoked excitatory postsynaptic potentials (EPSPs). NA-induced EPSP decrease was accompanied by effects on the presynaptic action potential (AP), reducing AP duration and amplitude of the after-hyperpolarization (AHP), without affecting the pre- and postsynaptic membrane potential. All effects of NA were blocked by yohimbine and synaptic transmission was reduced by clonidine, consistent with an action at presynaptic alpha 2-adrenoceptors. NA-induced inhibition of transmission was sensitive to pre-incubation of SCGNs with pertussis toxin (PTX), implicating the involvement of G alpha(i)/(o)beta y subunits. Expression of G alpha transducin, an agent which sequesters G protein beta gamma (G beta y) subunits, in the presynaptic neurone caused a time-dependent attenuation of NA-induced inhibition. Injection of purified G beta gamma subunits into the presynaptic neurone inhibited transmission, and also reduced the AHP amplitude. Furthermore, NA-induced inhibition was occluded by pre-injection of G beta gamma subunits. The Ca2+ channel blocker Cd2+ mimicked NA effects on transmitter release. Cd2+, NA and G beta gamma subunits also inhibited somatic Ca2+ current. In contrast to effects on AP-evoked transmitter release, NA had no clear action on AP-independent EPSPs induced by hypertonic solutions. These results demonstrate that G beta gamma subunits functionally mediate inhibition of transmitter release by alpha 2-adrenoceptors and represent important regulators of synaptic transmission at mammalian presynaptic terminals.

The relationship between membrane damage, release of protein and loss of viability in Escherichia coli exposed to high hydrostatic pressure

The aim of this work was to examine a possible association between resistance of two Escherichia coli strains to high hydrostatic pressure and the susceptibility of their cell membranes to pressure-induced damage. Cells were exposed to pressures between 100 and 700 MPa at room temperature (~20C) in phosphate-buffered-saline. In the more pressure-sensitive strain E. coli 8164, loss of viability occurred at pressures between 100 MPa and 300 MPa and coincided with irreversible loss of membrane integrity as indicated by uptake of propidium iodide (PI) and leakage of protein of molecular mass between 9 and 78 kDa from the cells. Protein release increased to a maximum at 400 MPa then decreased, possibly due to intracellular aggregation at the higher pressures. In the pressure-resistant strain E. coli J1, PI was taken up during pressure treatment but not after decompression indicating that cells were able to reseal their membranes. Loss of viability in strain J1 coincided with the transient loss of membrane integrity between approximately 200 MPa and 600 MPa. In E. coli J1 leakage of protein occurred before loss of viability and the released protein was of low molecular mass, between 8 and 11 kDa and may have been of periplasmic origin. In these two strains differences in pressure resistance appeared to be related to differences in the ability of their membranes to withstand disruption by pressure. However it appears that transient loss of membrane integrity during pressure can lead to cell death irrespective of whether cells can reseal their membranes afterwards.