硫胺素对Arthrobacter sp.A302环磷酸腺苷发酵的影响

研究在培养基中加入硫胺素（V B1）对cAMP发酵的影响。结果表明：V B1的最适添加量为0.5 g/L,与对照组相比,环磷酸腺苷（cAMP）产量和细胞干质量分别提高了36.4%和41.8%,达到7.5和7.8 g/L。琥珀酸和α-酮戊二酸的含量有明显的提高,平均提高了43.59%和40.77%;同时,主要副产物乙酸的含量没有明显变化。在发酵过程中,V B1的加入对ATP的含量及与cAMP合成密切相关的几种酶的活性也有显著的影响。     

Production of cyclic adenosine monophosphate by Arthrobacter sp. A302 using fed-batch fermentation with pH-shift control

The production of cyclic adenosine monophosphate (cAMP) by Arthrobacter sp. A302 was studied in a 5 L stirred tank fermentor under a range of pH values (6.5–8.0) and glucose feeding rates. In batch fermentation under a controlled pH, the optimum pH for cell growth was 7.5 with dry cell density (X) of 11.43 g L, and the optimum pH for cAMP accumulation was 7.0 with cAMP concentration of 7.41 g L. In order to achieve the high X and cAMP yield simultaneously, a pH-shift control strategy was proposed based on kinetic analysis of specific cell growth rate (μ) and specific cAMP formation rate (q _s ). In this method, pH was controlled to 7.0 for the first 30 h of fermentation, and then subsequently shifted to 7.5 and maintained until the end of the process. Application of this approach significantly enhanced the cAMP concentration. Thereafter, cAMP production was further improved by combining the above-mentioned pH-control system and fed-batch process with glucose at a constant feeding rate of 1.0 g L⁻¹ h⁻¹. Under optimum conditions, the final cAMP production was 10.87 g L, which is 110.0, 46.7, and 27.7% higher than that of the pH-uncontrolled, pH-controlled, and pH-shift controlled methods, respectively.     

Mediation of macrophage collagenase production by 3'-5' cyclic adenosine monophosphate

The production of collagenase by lipopolysaccharide-(LPS) activated guinea pig macrophages is mediated by prostaglandins (PG) of the E series. After stimulation of guinea pig macrophages with LPS, extracellular PGE levels and cellular cAMP levels are elevated. Indomethacin inhibits not only PG synthesis, but also cAMP and collagenase production in LPS-stimulated macrophage cultures. In these indomethacin-inhibited cultures containing LPS, dibutyryl (dB) cAMP, or cholera toxin can restore macrophage collagenase production but not PG synthesis. Moreover, dBcAMP and cholera toxin enhance collagenase production in LPS-activated cultures. Initial activation of the macrophages by an agent such as LPS is a prerequisite for synthesis of collagenase, since in the absence of LPS, dBcAMP or cholera toxin alone are ineffective stimuli. These findings clearly demonstrate a role for PG-induced elevations of cAMP in the production of collagenase by LPS-activated macrophages.     

发酵法生产L-异亮氨酸的溶氧控制策略

研究了溶氧对Brewibacterium lactofermentation分批发酵生产L-异亮氨酸（Ile）的影响,提出了前10h恒700d/min以维持溶氧在35％以上,10h后调至600r／min以维持溶氧在15％～20％的两阶段供氧控制模式。与对照相比,获得了较高的产率（0．094g／g）和糖耗速度（4．76/L·h）,在较短时间内（52h）获得较高的Ile产量（23．3g／L）,比结果最好的单一搅拌转速（600r/min）提高11．6％。生产强度（0．448d/L·h）比恒定搅拌转速（500、600、700、800r/min）控制下的过程分别提高了83．6％、28．7％、44．9％、35．7％。最后采用代谢通量分析对该结果产生的原因进行了定量解释。     

Fluid shear stress as a mediator of osteoblast cyclic adenosine monophosphate production

Effects of interstitial fluid flow on osteoblasts were investigated. Intracellular cyclic adenosine monophosphate (cAMP) levels were monitored in cultured osteoblasts subjected to shear rates ranging from 10 to 3,500 sec-1. Cyclic AMP levels were significantly increased at all shear rates from 1 pmole/mg protein to 10-16 pmole/mg protein. Osteoblasts subjected to a shear rate of 430 sec-1 for 0.5-15 minutes exhibited elevated levels (12-fold) of intracellular cAMP, which were sustained throughout the perfusion period. Osteoblasts were three times more sensitive to flow stimulation than human umbilical vein endothelial cells and baby hamster kidney fibroblasts, which also displayed higher cAMP levels (4-fold) after exposure to flow. To distinguish streaming potential effects from shear stress effects, viscosity was increased 5-fold by addition of neutral dextran to the perfusing medium. Shear stress is a function of viscosity, and streaming potentials are not for a given shear rate. The mechanism of this cellular response to flow was shown to be shear stress dependent. Inhibition of cyclooxygenase by 20 microM ibuprofen completely inhibited the flow-dependent cAMP response, indicating the cAMP response is mediated by prostaglandins. Our results suggest that fluid flow induced by mechanical stress may be an important mediator of bone remodeling.     

Regulation of phospholipid synthesis inMycobacterium smegmatis by cyclic adenosine monophosphate

Forskolin, an adenylate cyclase activator and a cyclic AMP analogue, dibutyryl cyclic AMP have been used to examine the relationship between intracellular levels of cyclic AMP and lipid synthesis inMycobacterium smegmatis. Total phospholipid content was found to be increased in forskolin grown cells as a result of increased cyclic AMP levels caused by activation of adenylate cyclase. Increased phospholipid content was supported by increased [¹⁴C] acetate incorporation as well as increased activity of glycerol-3-phosphate acyltransferase. Pretreatment of cells with dibutyryl cyclic AMP had similar effects on lipid synthesis. Taking all these observations together it is suggested that lipid synthesis is being controlled by cyclic AMP in mycobacteria.     

A melanin-dispersing effect of cyclic adenosine monophosphate on Fundulus melanophores

Treatment of Fundulus melanophores with adenosine 3′,5′-monophosphate (cyclic AMP) is followed by reversible melanin dispersion in these cells. Adenosine 3′-monophosphate and adenosine 5′-monophosphate both have a similar, but weaker dispersing action. In addition, adenosine 5′-monophosphate also has a melanin aggregating effect. These results are interpreted to mean that nerve transmitters may act by controlling the level of cyclic AMP within the Fundulus melanophore.     

The involvement of cyclic adenosine monophosphate in the control of schistosome miracidium cilia

This study examined the possible involvement of cyclic adenosine monophosphate (cAMP) in the control of ciliary action of Schistosoma mansoni miracidia. Miracidia immobilized in hypertonic NaCl solution were treated with 3 compounds that are known to increase intracellular cAMP concentrations. Forskolin, at a concentration of 50 microM, induced 50.1% of the miracidia to swim in hypertonic solution. The corresponding values obtained for 3-isobutyl-1-methylxanthine (IBMX) at 1 mM and 8-bromo-cAMP at 10 mM were 42.2 and 50.4%, respectively. The motility-enhancing effect of these compounds was dose dependent. Nevertheless, the swimming speed of miracidia activated in this way was only 10% of that observed in artificial pond water (APW). Cholera toxin had no apparent effect on miracidia swimming in hypertonic NaCl solution. Likewise, swimming in APW treated with forskolin at 50 microM, IBMX at 1 mM, or 8-bromo-cAMP at 10 mM did not induce any apparent change in motility. Miracidia swimming in APW were then treated with 3 compounds that decrease the intracellular concentration of cAMP. MDL-12,330A, at a concentration of 250 microM, caused a dramatic decrease in swimming over a period of 1 hr. Likewise, SQ22536 and imidazole, at concentrations of 20 and 50 mM, respectively, caused 36.5 and 73.4% decreases in swimming under the same conditions. Finally, inhibitors of cAMP-dependent protein kinase, i.e., PKI(14-22)amide, H89, and H88, completely inhibited miracidia swimming in APW at concentrations of 25, 50, and 100 microM, respectively. These results suggest that cAMP and cAMP-dependent protein kinase are involved in osmosis-controlled ciliary motion of schistosome miracidia.     

Studies of the cyclic adenosine monophosphate chemoreceptor ofParamecium

Summary A doublet of proteins (48,000M _r) from theParamecium cell body membrane fits several criteria for the external cAMP chemoreceptor. These criteria include: (i) selective elution from a cAMP affinity column, matching a specificity that could be predicted from the behavioral response and whole-cell binding; (ii) binding to wheat germ agglutinin indicating the presence of carbohydrate moieties indicating surface exposure; and (iii) selective inhibition of the intact cells' chemoresponse to cAMP by antibodies against the doublet. Additional evidence for the existence of a receptor, in general, comes from selective elimination of the cAMP chemoresponse by photoaffinity labeling of whole cells with 8-N₃-cAMP. The doublet proteins are not identical to the regulatory subunit of a cAMP-dependent protein kinase fromParamecium, theDictyostelium cAMP chemoreceptor, or the 42–45 kDa range proteins related to the large surface glycoprotein inParamecium. The doublet proteins are not readily separable and, as inDictyostelium, may represent two different covalent modification states of the same protein. Amino acid analysis indicates that the proteins are similar, but does not distinguish between the possibilities of proteolysis and covalent modification. Once cloned, this doublet may prove to be only the fifth external, eukaryotic chemoreceptor to be identified.     

Restoration of voltage-dependent antibrain antibody-inhibited calcium current by cyclic adenosine monophosphate

A dual-microelectrode voltage clamp technique was used for recording voltage-dependent calcium current (I_ca) in unidentified neurons isolated fromHelix pomatia. Neither intracellular injection of cyclic adenosine monophosphate (cAMP; 10 nA, 5 min) nor intracellular application of dibutyril-cAMP (dcAMP; 1 mM, 10–20 min) induced a change in normal I_ca or produce a reversible 10–20% reduction in amplitude. Adding S-100 protein fraction antibodies to the external medium led to the onset of calcium-dependent inactivation of I_ca, bringing amplitude of I_ca down to 15±12% of its initial level. Either cAMP or dcAMP then restored inhibited I_ca to 50±11% of its original level. It was found that the effects of cAMP on I_ca of intact neurons depend on level of cytoplasmic Ca²⁺.Institute for Brain Research, All-Union Center for Mental Health Research, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 21, No. 2, pp. 247–252, March–April, 1989.     

A calcium/calmodulin-dependent cyclic adenosine monophosphate phosphodiesterase from Drosophila heads

A Ca2+-activated cycl AMP phosphodiesterase from Drosophila melanogaster heads was studied. The enzyme accounted for approx. 40% of the total, soluble cyclic AMP phosphodiesterase activity in heads. After gel filtration, Ca2+ stimulation of the enzyme was no longer apparent, but Ca2+ activation could be restored by the addition of boiled Drosophila extract to the column-fractionated phosphodiesterase. The protein responsible for restoring Ca2+ activation was purified and shown to have some characteristics of calmodulin. In addition, porcine calmodulin was able to activate the Drosophila phosphodiesterase. Thus, the phosphodiesterase-calmodulin system in Drosophila appears analogous to similar systems in mammals.     

Dynamic mathematical models of batch experiments and fed-batch cultures for cyclic adenosine monophosphate production by <Emphasis Type="Italic">Arthrobacter</Emphasis> A302

A glucose utilizing strain, Arthrobacter A302 was used for cyclic adenosine monophosphate (cAMP) production in batch modes. The non-structured model in a 5 l stirred tank bioreactor for understanding, controlling, and optimizing the fermentation process was proposed using the logistic equation for microbial growth, the Luedeking-Piret equation for product formation and Luedeking-Piret-like equation for substrate uptake, respectively. The production of cAMP was a mixed-growth-associated pattern. Based on model prediction, a comparison of calculated value using the parameters evaluated above with another experimental data in 30 l bioreactor was used to test the model. The results predicted from the model were in good agreement with the experimental observations in 30 l bioreactor, which demonstrated that the model might be useful for the development and optimization of production of cAMP in industrial scale. Based on estimated kinetic parameters, three different fed-batch modes, constant rate and intermittent (once and repeated), were adopted in order to obtain more cAMP accumulation. Furthermore, the final production of cAMP reached 11.24 g l⁻¹ after 72 h incubation using three stages feeding strategy. In particular, the cAMP productivity (0.156 g l⁻¹ h⁻¹) was successfully improved by 22.83, 11.43 and 9.86%, respectively, compared with the modes of the batch, constant rate fed-batch and intermittent fed-batch once.     

Involvement of cyclic adenosine monophosphate in the control of motile behavior of Physarum polycephalum plasmodium

Possible involvement of autocrine factors into the control of motile behavior via a receptor-mediated mechanism was investigated in Physarum polycephalum plasmodium, a multinuclear amoeboid cell with the auto-oscillatory mode of motility. Cyclic adenosine monophosphate (cAMP) and extracellular cAMP-specific phosphodiesterase, its involvement into the control of plasmodium motile behavior was proved by action of its strong inhibitor, were regarded as putative autocrine factors. It was shown that the plasmodium secreted cAMP. When it was introduced into agar support, 0.1–1 mM cAMP induced a delay of the plasmodium spreading and its transition to migration. When locally applied, cAMP at the same concentrations induced the typical for attractant action increase in oscillation frequency and the decrease of ectoplasm elasticity. The ability to exhibit positive chemotaxis in cAMP gradient and the dependence of its realization were shown to depend on the plasmodium state. Chemotaxis test specimens obtained from the migrating plasmodium, unlike those obtained from growing culture, generate alternative fronts which compete effectively with fronts oriented towards the attractant increment. The results obtained support our supposition stated earlier that advance of the Physarum polycephalum plasmodium leading edge is determined by local extracellular cAMP gradients arising from a time delay between secretion and hydrolysis of the nucleotide.     

Modulation of postjunctional cholinergic sensitivity of rat diaphragm muscle by cyclic adenosine monophosphate

Summary Addition of 2.5 mM cyclic adenosine monophosphate (cAMP) to the solution bathing a rat diaphragm muscle alters the magnitude of depolarization responses to iontophoretic pulses of acetylcholine (ACh) at neuromuscular endplates. Alterations are repeatable with small variability on a given preparation for initial and repeat experiments on both hemidiaphragms, but are different on each preparation. Five min after addition of the nucleotide solution, increases (potentiations) of up to 30% above control levels and decreases (attenuations) to 50% below control levels are observed. The effects on sensitivity to ACh of dibutyryl cAMP (1.25 mM), monobutyryl cAMP (0.25 mM), and cAMP (2.5 mM) in Ca⁺⁺-free solution are a function of whether the experiment is an initial one on that preparation or a repeat experiment after 10 or more minutes of perfusion flow. In all three cases, initial exposure attenuates sensitivity (means at 5 min: –30, –10, and –20%, respectively) and repeat exposure potentiates sensitivity (means: 20% at 5 min, 15% at 5 min, and 10% at 2 min respectively). A concentration of dibutyryl cAMP (0.25 mM) which is without effect on sensitivity alone, produces a large, transient potentiation (mean: 45% at 1 min) in conjunction with 0.5 mM theophylline. A decrease in the rate of desensitization is observed during exposure to 0.25 mM cAMP. These results are interpreted in terms of a physiological mechanism whereby receptor activity at the postjunctional membrane is modulated by cAMP formed from prejunctionally released ATP.