Study of spatiotemporal regulation of kinase signaling using genetically encodable molecular tools

Precise spatiotemporal organization and regulation of signal transduction networks are essential for cellular response to internal and external cues. To understand how this biochemical activity architecture impacts cellular function, many genetically encodable tools which regulate kinase activity at a subcellular level have been developed. In this review, we highlight various types of genetically encodable molecular tools, including tools to regulate endogenous kinase activity and biorthogonal techniques to perturb kinase activity. Finally, we emphasize the use of these tools alongside biosensors for kinase activity to measure and perturb kinase activity in real time for a better understanding of the cellular biochemical activity architecture.     

Luminometric measurement of population activity of genetically modified Pseudomonas fluorescens in the soil

Genetically modified cells of Pseudomonas fluorescens, chromosomally marked with genes for bioluminescence, were inoculated into sterile soil microcosms. During incubation for 90 days, viable cell concentration did not change significantly but light output, measured by luminometry, decreased, indicating reduced metabolic activity due to lack of substrates. Amendment with nutrients resulted in parallel increases in both luminescence and dehydrogenase activity. Luminometry therefore enables rapid monitoring of the activity of populations of luminescence-marked microbial inocula in the soil, with greater sensitivity and selectivity than traditional techniques.     

Arginine kinase: Differentiation of gene expression and protein activity in the red imported fire ant, Solenopsis invicta

Arginine kinase (AK), a primary enzyme in cell metabolism and adenosine 5′-triphosphate (ATP)-consuming processes, plays an important role in cellular energy metabolism and maintaining constant ATP levels in invertebrate cells. In order to identify genes that are differentially expressed between larvae and adults, queens and workers, and female alates (winged) and queens (wingless), AK cDNA was obtained from the red imported fire ant. The cDNA sequence of the gene has open reading frames of 1065 nucleotides, encoding a protein of 355 amino acid residues that includes the substrate recognition region, the signature sequence pattern of ATP:guanidino kinases, and an “actinin-type” actin binding domain. Northern blot analysis and protein activity analysis demonstrated that the expression of the AK gene and its protein activity were developmentally, caste specifically, and tissue specifically regulated in red imported fire ants with a descending order of worker> alate (winged adult) female> alate (winged adult) male> larvae> worker pupae ≈ alate pupae. These results suggest a different demand for energy-consumption and production in the different castes of the red imported fire ant, which may be linked to their different missions and physiological activities in the colonies. The highest level of the AK gene expression and activity was identified in head tissue of both female alates and workers and thorax tissue of workers, followed by thorax tissue of female alates and abdomen tissue of male alates, suggesting the main tissues or cells in these body parts, such as brain, neurons and muscles, which have been identified as the major tissues and/or cells that display high and variable rates of energy turnover in other organisms, play a key role in energy production and its utilization in the fire ant. In contrast, in the male alate, the highest AK expression and activity were found in the abdomen, suggesting that here energy demand may relate to sperm formation and reproduction.     

Microtubule affinity regulating kinase activity in living neurons was examined by a genetically encoded fluorescence resonance energy transfer/fluorescence lifetime imaging-based biosensor: inhibitors with therapeutic potential

Protein kinases of the microtubule affinity regulating kinase (MARK)/Par-1 family play important roles in the establishment of cellular polarity, cell cycle control, and intracellular signal transduction. Disturbance of their function is linked to cancer and brain diseases, e.g. lissencephaly and Alzheimer disease. To understand the biological role of MARK family kinases, we searched for specific inhibitors and a biosensor for MARK activity. A screen of the ChemBioNet library containing ~18,000 substances yielded several compounds with inhibitory activity in the low micromolar range and capable of inhibiting MARK activity in cultured cells and primary neurons, as judged by MARK-dependent phosphorylation of microtubule-associated proteins and its consequences for microtubule integrity. Four of the compounds share a 9-oxo-9H-acridin-10-yl structure as a basis that will serve as a lead for optimization of inhibition efficiency. To test these inhibitors, we developed a cellular biosensor for MARK activity based on a MARK target sequence attached to the 14-3-3 scaffold protein and linked to enhanced cyan or teal and yellow fluorescent protein as FRET donor and acceptor pairs. Transfection of the teal/yellow fluorescent protein sensor into neurons and imaging by fluorescence lifetime imaging revealed that MARK was particularly active in the axons and growth cones of differentiating neurons.     

Biphenyl amide p38 kinase inhibitors 3: Improvement of cellular and in vivo activity

The biphenyl amides (BPAs) are a novel series of p38α MAP kinase inhibitor. The optimisation of the series to give compounds that are potent in an in vivo disease model is discussed. SAR is presented and rationalised with reference to the crystallographic binding mode.     

Effect of ferrous ion on the biological activity in a UASB reactor: mathematical modeling and verification

The effect of ferrous ion on the biological activity in a upflow anaerobic sludge blanket (UASB) reactor was studied. A mathematical model was developed and validated in order to simulate the dynamic behavior of a UASB reactor. This model took into consideration of all the biological and physicochemical reactions. The model was able to simulate the accumulation of iron in the sludge bed and its effect on the biological activity of the anaerobic sludge. A significant increase in the maximum uptake rate of methanogens and acidogens was revealed when iron was supplemented to the UASB reactor. The addition of ferrous iron induced a stable and excellent COD conversion rate. The model can be a useful tool for the prediction of process performance in the future and can be used to assist in the operation of biogas plants. The ferrous ion addition proved to enhance the biological activity of UASB sludge. Thus, the model can be used for the design of treatment plants that will take advantage of the benefits of iron addition.     

铰链结构对抗菌肽生物活性的影响及在分子设计中的应用研究进展

铰链结构,又称铰链区或转角,是部分抗菌肽序列中存在的一种特殊结构。但目前抗菌肽结构的研究多集中于标准的α-螺旋和β-折叠二级结构,对于铰链结构及其作用总结较少。铰链结构对抗菌肽生物活性有重要影响,主要原因是铰链结构能够提高抗菌肽的结构灵活性,促进其对细菌细胞膜的破坏作用或与胞内作用靶点的结合效率,进而提高抗菌肽的抗菌活性。同时,降低的抗菌肽结构刚性,消减了抗菌肽对真核细胞的毒性。文中结合了笔者课题组相关工作,就铰链结构特点、对抗菌肽生物活性的影响以及在抗菌肽分子设计方面的应用进行了综述,以期为新型抗菌肽的设计和开发提供参考。     

Survival-promoting activity of pituitary adenylate cyclase-activating polypeptide in the presence of phosphodiesterase inhibitors on rat motoneurons in culture: cAMP-protein kinase A-mediated survival

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to be neurotrophic or neuroprotective in various neurons in culture. It is expressed in spinal motoneurons in vivo and its expression is increased markedly after axotomy, suggesting a neuroprotective role via an autocrine mechanism. However, neurotrophic activity of PACAP has not been reported for motoneurons. In the present study, we investigated the effects of PACAP on rat motoneurons in culture. In the presence of a phosphodiesterase inhibitor, PACAP showed significant neurotrophic activity at concentrations as low as 0.01 nM. Previously, we found that glutamate was excitotoxic to motoneurons even in the presence of brain-derived neurotrophic factor, which is neurotrophic for motoneurons. PACAP with a phosphodiesterase inhibitor protected motoneurons against this excitotoxicity. The activity of PACAP was inhibited by the protein kinase A inhibitor N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide dihydrochloride, as was the case with the activity of forskolin, suggesting downstream involvement of a cAMP-protein kinase A signaling pathway. The present results may suggest a physiological role of PACAP in vivo, and implicate the PACAP-cAMP signaling pathway for the possible therapeutic target of amyotrophic lateral sclerosis as glutamate excitotoxicity was suggested in sporadic amyotrophic lateral sclerosis.     

Oxidative stress in biological systems and its relation with pathophysiological functions: the effect of physical activity on cellular redox homeostasis

The body of evidence from the past three decades demonstrates that oxidative stress can be involved in several diseases. This study aims to summarise the current state of knowledge on the association between oxidative stress and the pathogenesis of some characteristic to the biological systems diseases and aging process. This review also presents the effect of physical activity on redox homeostasis. There is strong evidence from studies for participation of reactive oxygen and nitrogen species in pathogenesis of acute and chronic diseases based on animal models and human studies. Elevated levels of pro-oxidants and various markers of the oxidative stress and cells and tissues damage linked with pathogenesis of cancer, atherosclerosis, neurodegenerative diseases hypertension, diabetes mellitus, cardiovascular disease, atherosclerosis, reproductive system diseases, and aging were reported. Evidence confirmed that inflammation contributes widely to multiple chronic diseases and is closely linked with oxidative stress. Regular moderate physical activity regulates oxidative stress enhancing cellular antioxidant defence mechanisms, whereas acute exercise not preceded by training can alter cellular redox homeostasis towards higher level of oxidative stress. Future studies are needed to clarify the multifaceted effects of reactive oxygen/nitrogen species on cells and tissues and to continue study on the biochemical roles of antioxidants and physical activity in prevention of oxidative stress-related tissue injury.     

FRET-based direct detection of dynamic protein kinase A activity on the sarcoplasmic reticulum in cardiomyocytes

The second messenger cAMP-dependent protein kinase A (PKA) plays an important role in the various cellular and physiological responses. On the sarcoplasmic reticulum (SR) in cardiomyocytes, PKA regulates the calcium cycling for exciting–contraction coupling, which is often dysfunctional in a variety of heart diseases including heart failure. Here, we have developed a novel FRET-based A-kinase activity biosensor (AKAR), termed SR-AKAR3, to visualize the PKA dynamics on the SR. Activation of adrenergic receptor induces a rapid and significant increase in SR-AKAR3 FRET ratio, which is dependent on agonist occupation of the receptor and inhibited by H-89, a PKA inhibitor. Interestingly, direct activation of adenylyl cyclases or application of a cAMP analog 8-Br-cAMP induced much slower and smaller increases in SR-AKAR3 FRET ratio. These data indicate that the signaling induced by adrenergic stimulation displays a preferential access to the SR in comparison to those by direct activation of adenylyl cyclases. More, SR-AKAR3 mimics endogenous protein phospholamban on the SR for PKA-mediated phosphorylation and myocyte contraction response under adrenergic stimulation. Together, this new PKA activity biosensor provides a useful tool to directly visualize the dynamic regulation of PKA activity on the SR in cardiomyocytes under various physiological and clinical conditions.     

Effects of parental and progeny rearing densities on locomotor activity of 1st-stadium nymphs in the migratory locust, Locusta migratoria: An analysis by long-term monitoring using an actograph

The effects of parental and progeny rearing densities on locomotor activity in 1st-stadium nymphs of the migratory locust, Locusta migratoria, were observed over a 24- or 36-h period using an actograph. Newly hatched nymphs showed a small activity peak shortly after hatching and the peak level was significantly higher in offspring (gregarious nymphs) of crowd-reared adults than in those (solitarious nymphs) of isolated-reared adults. However, no significant difference was found between the two groups in maximum activity levels exhibited after the initial peak. Post-hatching crowding enhanced locomotor activity during 2-5 h of measurements in 2-day-old nymphs. In this case, the parental density resulted in no significant influence on locomotor activity. However, the maximum activity level shown later in the observation period was higher in gregarious nymphs than in solitarious nymphs. Interestingly, this parental effect was more pronounced in nymphs reared in group than in those reared in isolation. The parental density appeared to affect the degree of response to crowding in the progeny. No evidence was found for the phase accumulation in terms of locomotor activity. The variation observed in locomotor activity among geographical populations did not correspond to their phylogenetic relationships.