Amplification of pulsatile glucagon counterregulation by switch-off of alpha-cell-suppressing signals in streptozotocin-treated rats

Glucagon counterregulation (GCR) is a key protection against hypoglycemia that is compromised in diabetes via an unknown mechanism. To test the hypothesis that alpha-cell-inhibiting signals that are switched off during hypoglycemia amplify GCR, we studied streptozotocin (STZ)-treated male Wistar rats and estimated the effect on GCR of intrapancreatic infusion and termination during hypoglycemia of saline, insulin, and somatostatin. Times 10 min before and 45 min after the switch-off were analyzed. Insulin and somatostatin, but not saline, switch-off significantly increased the glucagon levels (P = 0.03), and the fold increases relative to baseline were significantly higher (P < 0.05) in the insulin and somatostatin groups vs. the saline group. The peak concentrations were also higher in the insulin (368 pg/ml) and somatostatin (228 pg/ml) groups vs. the saline (114 pg/ml) group (P < 0.05). GCR was pulsatile in most animals, indicating a feedback regulation. After the switch-off, the number of secretory events and the total pulsatile production were lower in the saline group vs. the insulin and somatostatin groups (P < 0.05), indicating enhancement of glucagon pulsatile activity by insulin and somatostatin compared with saline. Network modeling analysis demonstrates that reciprocal interactions between alpha- and delta-cells can explain the amplification by interpreting the GCR as a rebound response to the switch-off. The model justifies experimental designs to further study the intrapancreatic network in relation to the switch-off phenomenon. The results of this proof-of-concept interdisciplinary study support the hypothesis that GCR develops as a rebound pulsatile response of the intrapancreatic endocrine feedback network to switch-off of alpha-cell-inhibiting islet signals.     

Changes in amino acid and nucleotide pools of Rhodospirillum rubrum during switch-off of nitrogenase activity initiated by NH4+ or darkness.

Amino acid and nucleotide pools were measured in nitrogenase-containing Rhodospirillum rubrum cultures during NH4+- or dark-induced inactivation (switch-off) of the Fe protein. A big increase in the glutamine pool size preceded NH4+ switch-off of nitrogenase activity, but the glutamine pool remained unchanged during dark switch-off. Furthermore, methionine sulfoximine had no effect on the rate of dark switch-off, suggesting that glutamine plays no role in this process. In the absence of NH4+ azaserine, an inhibitor of glutamate synthate, raised glutamine pool levels sufficiently to initiate switch-off in vivo. While added NH4+ substantially increased the size of the nucleotide pools in N-limited cells, the kinetics of nucleotide synthesis were all similar and followed (rather than preceded) Fe protein inactivation. Darkness had little effect on nucleotide pool sizes. Glutamate pool sizes were also found to be important in NH4+ switch-off because of the role of this molecule as a glutamine precursor. Much of the diversity reported in the observations on NH4+ switch-off appears to be due to variations in glutamate pool sizes prior to the NH4+ shock. The nitrogen nutritional background is an important factor in determining whether darkness initiates nitrogenase switch-off; however, no link has yet been established between this and NH4+ (glutamine) switch-off.     

Effect of an ntrC mutation on amino acid or urea utilization and on nitrogenase switch-off in Herbaspirillum seropedicae

Herbaspirillum seropedicae is a nitrogen-fixing bacterium that grows well with ammonium chloride or sodium nitrate as alternative single nitrogen sources but that grows more slowly with L-alanine, L-serine, L-proline, or urea. The ntrC mutant strain DCP286A was able to utilize only ammonium or urea of these nitrogen sources. The addition of 1 mmol.L-1 ammonium chloride to the nitrogen-fixing wild-type strain inhibited nitrogenase activity rapidly and completely. Urea was a less effective inhibitor; approximately 20% of nitrogenase activity remained 40 min after the addition of 1 mmol x L-1 urea. The effect of the ntrC mutation on nitrogenase inhibition (switch-off) was studied in strain DCP286A containing the constitutively expressed gene nifA of H. seropedicae. In this strain, nitrogenase inhibition by ammonium was completely abolished, but the addition of urea produced a reduction in nitrogenase activity similar to that of the wild-type strain. The results suggest that the NtrC protein is required for assimilation of nitrate and the tested amino acids by H. seropedicae. Furthermore, NtrC is also necessary for ammonium-induced switch-off of nitrogenase but is not involved in the mechanism of nitrogenase switch-off by urea.     

Ammonia switch-off of nitrogenase from Rhodobacter sphaeroides and Methylosinus trichosporium: no evidence for Fe protein modification

In vivo switch-off of nitrogenase activity by NH ₄ ⁺ is a reversible process in Rhodobacter sphaeroides and Methylosinus trichosporium OB3b. The same pattern of switch-off in Rhodospirillum rubrum is explained by ADP-ribosylation of one of the Fe protein subunits, however, no evidence of covalent modification could be found in the subunits from either R. sphaeroides or M. trichosporium. Fe protein subunits from these organisms showed no variant behaviour on SDS-PAGE, nor were they ³²P-labeled following switch-off. These observations suggest either that the attachment of the modifying group to the Fe protein in these organisms is quite labile and does not survive in vitro manipulation, or that the mechanism of switch-off is different than that seen in Rhodospirillum.     

Preference and aversion for brain stimulations estimated by the conditioned place preference

Rats implanted with chronic electrode into the medial forebrain bundle at the level of the hypothalamus (which elicited self-stimulation behaviour) display conditioned place preference after repeated stimulations of this area; conversely rats implanted into the mesencephalic dorso-medial tegmentum (which elicited switch-off behaviour) present conditioned place aversion after such repeated stimulations. Furthermore rats implanted in these two area without exhibiting self-stimulation or switch-off behaviours also display preference or aversion for the location paired with the brain stimulations. It was thus hypothesized that the conditioned place preference procedure seemed to present a higher sensitivity than the bar pressing procedures to detect preference or aversion for brain stimulation. Moreover the activation of the medial forebrain bundle which was associated with positive affect is more effective for a long term retention of the preferred location than midbrain periventricular reticular activation which induces a negative affect.     

酰胺态氮瞬间调节荚膜红假单孢菌光合固氮活性的GS传感机制

天门冬酰胺(Asn)和谷氨酰胺(Gln)对荚膜红假单孢菌固氮酶活性抑制,在表观上类似于氨关闭效应,这种抑制效应由GS参与,相似于氨抑的传感机制。中断Gln代谢的6-diazo-5-oxo-L-norleucine(DON)存在时,氨抑的持续时间延长,与此相类似,Gln抑制加剧,这可能归之于Gln的积累。但是,Gln抑制被methionine sulfoximine(MSX,GS的抑制剂)消除,消除时MSX对Gln的浓度比值约为0.2,与氨抑消除所需的MSX对氨的浓度比值相当。此外,MSX消除氨抑不为DON拮抗,表明Gln抑制固氮酶活性由GS传感。然而,不能抑制GS转谷酰基活性的methionine suffone(MSF,谷氨酸的类似物)却与MSX相同,能消除Gln和氨对固氮活性的抑制。上述观察结果也可延伸至Asn的关闭固氮酶活性效应。     

Studies of the adenylate and pyridine nucleotide pools during nitrogenase ‘switch-off’ inRhodospirillum rubrum

Summary When ammonium ions are added to a nitrogen fixing culture ofRhodospirillum rubrum, nitrogenase activity decreases due to inactivation of the Fe-protein. We have studied the adenylate and pyridine nucleotide pools during switch-off using the sensitive bioluminescence method. Immediately after the addition of ammonium ions there is a decrease in the ATP pool which is quickly reversed and no change is seen during the switch-off period. The pyridine nucleotide pools also do not change significantly during the switch-off. Consequently we conclude that changes in the pools studied were not the signal promoting inactivation of the Fe-protein.     

2-Oxoglutarate and the PII homologues NifI1 and NifI2 regulate nitrogenase activity in cell extracts of Methanococcus maripaludis

Summary Post-translational regulation of nitrogen fixation, or switch-off, in the methanogenic archaeon Methanococcus maripaludis does not involve detectable covalent modification of the dinitrogenase reductase as in some bacteria, and the genes encoding the PII homologues NifI(1) and NifI(2) are both required, indicating a novel mechanism. To further understand the mechanism of switch-off, we assayed nitrogenase activity in cell extracts from wild-type and nifI mutant strains in the absence or presence of potential signals of nitrogen status. Activity in extracts from a DeltanifI(1)nifI(2) strain was sixfold higher than in extracts from wild-type cells. Addition of 2-oxoglutarate to wild-type extracts enhanced activity up to fivefold, a level similar to that observed in DeltanifI(1)nifI(2) extracts. 2-Oxoglutarate did not affect activity in DeltanifI(1)nifI(2) or single nifI mutant extracts. Furthermore, extracts from genetically complimented nifI mutants regained wild-type characteristics, indicating an in vitro correlation with in vivo effects. Extraction and quantification of 2-oxoglutarate indicated concentrations 10-fold higher in nitrogen-fixing cells than in switched-off and ammonium-grown cells. We propose a model for switch-off where the NifI proteins have an inhibitory effect on nitrogenase activity that is counteracted by high levels of 2-oxoglutarate, which acts as a signal of nitrogen limitation.     

Uridylylation of the P(II) protein in the photosynthetic bacterium Rhodospirillum rubrum.

The regulatory protein P(II) has been studied in great detail in enteric bacteria; however, its function in photosynthetic bacteria has not been clearly established. As a number of these bacteria have been shown to regulate nitrogenase activity by a metabolic control system, it is of special interest to establish the role of P(II) in these diazotrophs. In this study, we show that P(II) in Rhodospirillum rubrum is modified in response to the N status in the cell and that addition of ammonium or glutamine leads to demodification. We also provide evidence that P(II) is uridylylated. In addition, we show that not only these compounds but also NAD+ promotes demodification of P(II), which is of particular interest as this pyridine nucleotide has been shown to act as a switch-off effector of nitrogenase. Demodification of P(II) by ammonium or NAD+ did not occur in cultures treated with an inhibitor of glutamine synthetase (methionine sulfoximine), whereas treatment with the glutamate synthase inhibitor 6-diazo-5-oxo-norleucine led to total demodification of P(II) without any other addition. The results indicate that P(II) probably is not directly involved in darkness switch-off of nitrogenase but that a role in ammonium switch-off cannot be excluded.     

The mating-induced refractoriness of Lucilia cuprina females: manipulating the male contribution

Abstract Three manipulations of the male contribution to copulation were used to investigate what determines the switch-off of female receptivity that follows mating in Lucilia cuprina. Multiple matings by males led to a reduction in their effectiveness at switching-off females: the first twelve matings caused more than 50% of females to be switched-off 1 day after mating, while the first four matings caused more than 50% of females to be switched-off for 7 days after mating. Males mated up to twenty-three times and on average ten times during 10 h of continuous access to virgin females. The number of sperm transferred declined logarithmically through a series of matings as did the quantity of material transferred from the male accessory gland (measured as radiolabeled material) through the first six matings. Overnight isolation of multiply-mated males led to considerable recovery of their ability to switch-off females, and to limited renewal of their ability to transfer sperm. Castrated males transferred similar quantities of accessory gland material to females as did sham-operated males during their first five matings and in their first three matings switched-off 95% of females for 1 day but only 48% for 7 days. When normal mating pairs were separated at increasing intervals after coupling, an increasing proportion of females were switched-off at 1 or 7 days afterward. 7 days after 2 min matings no females were switched-off though 40% of the number of sperm transferred in a full mating had been transferred by this time. The proposed explanation for these data is that both the initial switch-off and its duration are determined by the quantity of a receptivity-inhibiting substance that normally enters the female haemo-lymph after being injected by the male into the wall of the bursa copulatrix. It is proposed that when castrated males mate, an absence of sperm results in most accessory gland secretion entering the empty spermathecae (rather than the wall of the bursa as usually occurs) and hence being unable to reach the haemolymph and exert its influence. The effective dose of the receptivity-inhibiting substance is measured on a logarithmic scale.     

Regulatory properties of the nitrogenase fromRhodopseudomonas palustris

Ammonium salts, glutamine, asparagine, and urea cause an immediate inactivation (switch-off) of light-dependent acetylene reduction in intact cells of the photosynthetic bacteriumRhodopseudomonas palustris. This effect is reversible showing the same kinetic pattern of inactivation and reactivation with all effector compounds. Its duration depends on the amount of effector added to the cells. Both nitrogenase components are found catalytically active in a cell-free preparation after enzyme switch-off in vivo. Involvement of the ammonia assimilating system in this regulatory mechanism is indicated by the following observations: ammonia uptake during the switch-off period, resumption of acetylene reduction after disappearance of ammonia from the outer medium, and persistence of enzyme switch-off with dihydrogen and thiosulfate as electron donors in the absence of an additional carbon source. Nitrogenase activity in crude extracts is non-linear with time and is stimulated by manganese ions. After resolution of nitrogenase into its MoFe-protein and Fe-protein these properties are lost, indicating the presence of an activating factor. Nitrogenase ofR. palustris cross reacts reciprocally with the complementary proteins ofAzotobacter vinelandii, but not with those ofClostridium pasteurianum.Abbreviations CCCP m-chlorocarbonyl cyanide phenyl hydrazone - DNP 2,4-dinitrophenol - EPR electron paramagnetic resonance - HEPES N-2-hydroxyethylpiperazine--2-ethane sulfonic acid - NOQNO 2-n-nonyl-4-hydroxyquinoline-N-oxide - TES N-tris[hydroxymethyl]methyl-2-aminoethane sulfonic acid     

Control of Plasmid R1 Replication: Functions Involved in Replication, Copy Number Control, Incompatibility, and Switch-off of Replication

A small derivative of plasmid R1 was used to integratively suppress a chromosomal dnaA(Ts) mutation. The strain obtained grew normally at 42°C. The integratively suppressed strain was used as recipient for various plasmid R1 derivatives. Plasmid R1 and miniplasmid derivatives of R1 could be established in the strain that carried an integrated R1 replicon, but they were rapidly lost during growth. However, plasmids also carrying ColE1 replication functions were almost completely stably inherited. The integratively suppressed strain therefore allows the establishment of bacteria diploid with respect to plasmid R1 and forms a useful and sensitive system for studies of interaction between plasmid R1 replication functions. Several of the chimeric plasmids caused inhibition of growth at high temperatures. All plasmids that inhibited growth carried one particular PstI fragment from plasmid R1 (the PstI F fragment), and in all cases the growth inhibition could be ascribed to repression of initiation of chromosome replication at 42°C, i.e., they carry a trans-acting switch-off function. Furthermore, the analogous PstI fragments from different copy mutants of plasmid R1 were analyzed similarly, and one mutant was found to lack the switch-off function. The different chimeric plasmids were also tested for their incompatibility properties. All plasmids that carried the switch-off function (and no other plasmids) also carried R1 incompatibility gene(s). Since the PstI F fragment, which is present on all these plasmids, is very small (0.35 × 10⁶), it is suggested that the switch-off regulation of replication (by an inhibitor), incompatibility, and copy number control are governed by the same gene.     

L-Methionine-SR-sulfoximine as a probe for the role of glutamine synthetase in nitrogenase switch-off by ammonia and glutamine in Rhodopseudomonas palustris

Methionine sulfoximine (MSX), an irreversible inhibitor of glutamine synthetase of Rhodopseudomonas palustris restored nitrogenase activity to cells in which nitrogenase had been completely inhibited by ammonia switch-off. After addition of MSX, there was a lag period before nitrogenase activity was fully restored. During this lag, glutamine synthetase activity progressively decreased, and near the time of its complete inhibition, nitrogenase activity resumed. Nitrogenase switch-off by ammonia thus required active glutamine synthetase. Glutamine itself caused nitrogenase inhibition whose reversal by MSX depended on the relative ratio of MSX to glutamine. Unlike ammonia, glutamine inhibited nitrogenase under conditions where glutamine synthetase activity was absent. This indicates that glutamine is the effector molecule in nitrogenase switch-off, for instance by interacting with the enzymatic system for Fe protein inactivation. The effects of glutamine and MSX were also dependent on the culture age. Possible explanation for this and for the competitive effects are a common binding site within the regulatory apparatus for nitrogenase, or, in part, within a common transport system. Some observations with MSX were extended to Rhodopseudomonas capsulata and agreed with those in R. palustris.     

Reversible membrane association of dinitrogenase reductase activating glycohydrolase in the regulation of nitrogenase activity in Rhodospirillum rubrum; dependence on GlnJ and AmtB1

In the photosynthetic bacterium Rhodospirillum rubrum nitrogenase activity is regulated by reversible ADP-ribosylation of dinitrogenase reductase in response to external so called "switch-off" effectors. Activation of the modified, inactive form is catalyzed by dinitrogenase reductase activating glycohydrolase (DRAG) which removes the ADP-ribose moiety. This study addresses the signal transduction between external effectors and DRAG. R. rubrum, wild-type and P(II) mutant strains, were studied with respect to DRAG localization. We conclude that GlnJ clearly has an effect on the association of DRAG to the membrane in agreement with the effect on regulation of nitrogenase activity. Furthermore, we have generated a R. rubrum mutant lacking the putative ammonium transporter AmtB1 which was shown not to respond to "switch-off" effectors; no loss of nitrogenase activity and no ADP-ribosylation. Interestingly, DRAG was mainly localized to the cytosol in this mutant. Overall the results support our model in which association to the membrane is part of the mechanism regulating DRAG activity.     

Nitrogenase switch-off by ammonium ions in Azospirillum brasilense requires the GlnB nitrogen signal-transducing protein

Nitrogenase activity in several diazotrophs is switched off by ammonium and reactivated after consumption. The signaling pathway to this system in Azospirillum brasilense is not understood. We show that ammonium-dependent switch-off through ADP-ribosylation of Fe protein was partial in a glnB mutant of A. brasilense but absent in a glnB glnZ double mutant. Triggering of inactivation by anaerobic conditions was not affected in either mutant. The results suggest that glnB is necessary for full ammonium-dependent nitrogenase switch-off in A. brasilense.     

刺激黄雀新纹状体前部大细胞核外侧部对发声和呼吸的影响

实验材料为乌拉坦麻醉的鸣禽黄雀（Ｃａｒｄｕｅｌｉｓ ｓｐｉｎｕｓ）。观察了电及化学刺激新纹状体前部大细胞核外侧部（１ＭＡＮ）对发声和呼吸的影响。结果如下：（１）电刺激１ＭＡＮ的不同区域都引起鸣叫反应。（２）长串电脉冲刺激１ＭＡＮ使呼吸频率增加,呼吸幅度降低。（３）短串电脉冲刺激１ＭＡＮ,落位于吸气相,产生吸气要断效应;落位于呼气相,可使呼气时程延长,以冲刺激１ＭＡＮ,落位于吸气相,产生吸气切断效应     

Role of proton motive force in the infection of E. coli K-12 cells by bacteriophage T4]

It was shown that infection of E. coli cells by phage T4 is suppressed, when the cells are treated by oxidative phosphorylation uncouplers. The inhibiting effects of the uncouplers manifest themselves at the stage of phage DNA entry into the cells. Study of the E. coli cells devoid of their H+-ATPase activity due to mutation showed that the infection is suppressed by a switch-off of the respiratory chain, the only generator of the proton motive force (PMF) in mutated cells. Infection of the E. coli cells containing intact H+-ATPase occured even in the case when the respiratory chain activity was inhibited. The kinetic studies showed that generation of PMF is necessary during phage DNA transport into the cells and is indispensable for phage DNA entry into bacterial cells.