Low ATP Concentrations Research Articles

Mixomics analysis of Bacillus subtilis: effect of oxygen availability on riboflavin production

BackgroundRiboflavin, an intermediate of primary metabolism, is one kind of important food additive with high economic value. The microbial cell factory Bacillus subtilis has already been proven to possess significant importance for the food industry and have become one of the most widely used riboflavin-producing strains. In the practical fermentation processes, a sharp decrease in riboflavin production is encountered along with a decrease in the dissolved oxygen (DO) tension. Influence of this oxygen availability on riboflavin biosynthesis through carbon central metabolic pathways in B. subtilis is unknown so far. Therefore the unveiled effective metabolic pathways were still an unaccomplished task till present research work.ResultsIn this paper, the microscopic regulation mechanisms of B. subtilis grown under different dissolved oxygen tensions were studied by integrating 13C metabolic flux analysis, metabolomics and transcriptomics. It was revealed that the glucose metabolic flux through pentose phosphate (PP) pathway was lower as being confirmed by smaller pool sizes of metabolites in PP pathway and lower expression amount of ykgB at transcriptional level. The latter encodes 6-phosphogluconolactonase (6-PGL) under low DO tension. In response to low DO tension in broth, the glucose metabolic flux through Embden–Meyerhof–Parnas (EMP) pathway was higher and the gene, alsS, encoding for acetolactate synthase was significantly activated that may result due to lower ATP concentration and higher NADH/NAD+ ratio. Moreover, ResE, a membrane-anchored protein that is capable of oxygen regulated phosphorylase activity, and ResD, a regulatory protein that can be phosphorylated and dephosphorylated by ResE, were considered as DO tension sensor and transcriptional regulator respectively.ConclusionsThis study shows that integration of transcriptomics, 13C metabolic flux analysis and metabolomics analysis provides a comprehensive understanding of biosynthesized riboflavin’s regulatory mechanisms in B. subtilis grown under different dissolved oxygen tension conditions. The two-component system, ResD–ResE, was considered as the signal receiver of DO tension and gene regulator that led to differences between biomass and riboflavin production after triggering the shifts in gene expression, metabolic flux distributions and metabolite pool sizes.

Kca3.1 Activation Via P2y2 Purinergic Receptors Promotes Human Ovarian Cancer Cell (Skov-3) Migration

Disorders in cell signaling mediated by ATP or histamine, activating specific membrane receptors, have been frequently associated with tumorigenesis. Among the elements of response to purinergic (and histaminergic) signaling, ion channel activation controls essential cellular processes in cancer, such as cell proliferation, motility, and death. Here, we studied the effects that ATP had on electrical properties of human ovarian adenocarcinoma cells named SKOV-3. ATP caused increase in intracellular Ca2+ concentration ([Ca2+]i) and, concurrently, it evoked a complex electrical response with a conspicuous outward component. This current was generated through P2Y2 receptor activation and opening of K+ channels, KCa3.1, as indicated by electrophysiological and pharmacological analysis, as well as by immunodetection and specific silencing of P2Y2 or KCa3.1 gene by esiRNA transfection. Low µM ATP concentration increased SKOV-3 cell migration, which was strongly inhibited by KCa3.1 channel blockers and by esiRNA-generated P2Y2 or KCa3.1 downregulation. Finally, in human ovarian tumors, the P2Y2 and KCa3.1 proteins are expressed and co-localized in neoplastic cells. Thus, stimulation of P2Y2 receptors expressed in SKOV-3 cells promotes motility through KCa3.1 activation. Since P2Y2 and KCa3.1 are co-expressed in primary tumors, our findings suggest that they may play a role in cancer progression.

Pnma5 is essential to the progression of meiosis in mouse oocytes through a chain of phosphorylation.

PNMA (paraneoplastic antigen MA) family includes Pnma1–6. Although other members have been found to be involved in paraneoplastic neurological disorders, death receptor-dependent apoptosis, and tumorigenesis, Pnma5 was thought to be a female fertility factor, as indicated by one genome-wide study. But until now there have not been any further functional studies about Pnma5 in female meiosis. Our preliminary study indicated that Pnma5 might play important roles in meiosis. To further address this, Pnma5 was knocked down in in-vitro maturated (IVM) mouse oocytes, which are common models for mammalian female meiosis, by specific siRNA, and results showed that the loss of Pnma5 significantly delayed the progression of meiosis I and increased chromosome segregation errors during anaphase I. In in-vitro fertilization (IVF), Pnma5 knockdown caused significantly lower fertilization. To assess how it affects meiosis, Pnma5 knockdown was found to significantly decrease the stability of spindle microtubules and altered F-actin organization within actin cap regions, cause significantly abnormal mitochondria aggregation and lower ATP concentration. Next we have found that phosphorylation at Thr533 re-located Pnma5 strongly to spindles & cortex and was required for the phosphorylation of Akt and Gsk3β, while Src and Erk1/2 phosphorylation was required for the phosphorylation of Pnma5, indicating that phosphorylated Pnma5 is the active form and subsequently activates Akt and Gsk3β. Collectively this study suggests that Pnma5 is important for meiosis and is the pivot of Src→Erk1/2→Pnma5→Akt→Gsk3β pathway.

Free Trehalose Accumulation in Dormant Mycobacterium smegmatis Cells and Its Breakdown in Early Resuscitation Phase.

Under gradual acidification of growth medium resulting in the formation of dormant Mycobacterium smegmatis, a significant accumulation of free trehalose in dormant cells was observed. According to 1H- and 13C-NMR spectroscopy up to 64% of total organic substances in the dormant cell extract was represented by trehalose whilst the trehalose content in an extract of active cells taken from early stationary phase was not more than 15%. Trehalose biosynthesis during transition to the dormant state is provided by activation of genes involved in the OtsA-OtsB and TreY-TreZ pathways (according to RT-PCR). Varying the concentration of free trehalose in dormant cells by expression of MSMEG_4535 coding for trehalase we found that cell viability depends on trehalose level: cells with a high amount of trehalose survive much better than cells with a low amount. Upon resuscitation of dormant M. smegmatis, a decrease of free trehalose and an increase in glucose concentration occurred in the early period of resuscitation (after 2 h). Evidently, breakdown of trehalose by trehalase takes place at this time as a transient increase in trehalase activity was observed between 1 and 3 h of resuscitation. Activation of trehalase was not due to de novo biosynthesis but because of self-activation of the enzyme from the inactive state in dormant cells. Because, even a low concentration of ATP (2 mM) prevents self-activation of trehalase in vitro and after activation the enzyme is still sensitive to ATP we suggest that the transient character of trehalase activation in cells is due to variation in intracellular ATP concentration found in the early resuscitation period. The negative influence of the trehalase inhibitor validamycin A on the resuscitation of dormant cells proves the importance of trehalase for resuscitation. These experiments demonstrate the significance of free trehalose accumulation for the maintenance of dormant mycobacterial viability and the involvement of trehalose breakdown in early events leading to cell reactivation similar to yeast and fungal spores.

The Power Stroke Distance of Human Cytoplasmic Dynein

Cytoplasmic dynein is a motor protein moving along microtubules, and plays important roles in vesicle transport and mitosis. To understand the molecular mechanism of dynein motility, we measured the efficiency of FRET from dynein ring-BFP to linker-GFP, and the displacement driven by single-headed dynein interacting with microtubules by optical tweezers. The efficiency and the apparent stroke displacement depend on ATP concentration. The low efficiency and displacement at low ATP concentration indicate no conformational change of dynein at no ATP binding (apo) state that is predominant at low ATP concentration. With increasing ATP concentration, population of apo state decreases and that of the pre-power stroke state such as dynein ADP-Pi should increase. Dynein at the pre-power stroke state will generate the power stroke at binding to microtubule. Therefore, the displacement increased with ATP concentration. This is the first obtained interesting result and has not been reported for myosin and kinesin power strokes. High FRET efficiency and distance (8-9 nm) at saturated concentration of ATP indicate that the 8-9 nm power stroke distance is generated by swing of linker. This is supported by the result that the dynein deleted at the loops interacted with the linker did not generate the power stroke. The power stroke driven by the conformational change of dynein linker will be the fundamental mechanism of dynein motility.

Study of Bloom resolving G-quadruplex process by using high resolution magnetic tweezer with illumination of total internal reflection

G-quadruplex (G4) is a DNA structure which commonly exists in human genome, and it is considered as an important structure in DNA metabolism such as replication, transcription and homologous recombination. The G-quadruplex helicases have been widely investigated these years. Of them, the Bloom (BLM) helicase is most thoroughly studied. However, there are some basic problems that are still unclear. Most of previous studies of G4 are performed by single molecule fluorescence resonance energy transfer technique. The G4 is in a free state in these experiments, which is different from the physiological environment in cells. The traditional magnetic tweezers have a limitation of spatial resolution in a low force circumstance. Thus here we use high resolution magnetic tweezer under the illumination of total internal reflection fluorescence to study the process of BLM resolving G4. Our modification of magnetic tweezer is to separate the measurements of force and distance of magnetic tweezer in order to improve the spatial resolution, which allows us to observe the unfolding process of G4. With a 2-3 pN force we find that the process of BLM unfolding G4 in low ATP concentration is stepwise, and the G4 is mainly in the state between G-quadruplex and G-triplex. We also find that the BLM could interact with G4 for a long time. Our apparatus is also able to obtain the long time observation results compared with the single molecule fluorescence technique. So we perform experiments with a nearly saturated ATP concentration. We find that the BLM has two ways to maintain G4 dissolution in this condition. The BLM could unfold the G4 repetitively in a long period and it could also keep the G4 in unfolding state for a long time after it has opened the G4. Finally, we also perform single molecule fluorescence resonance energy transfer experiment in the same condition, and we find that the 2-3 pN force in magnetic tweezers has a rare influence on the process of BLM interacting with G4. The results of single molecule fluorescence resonance energy transfer experiments are corresponding to the results of magnetic tweezer in the same conditions. All of our experimental results show that ATP dependent BLM has a high affinity with G4 and BLM has a different way to resolve G4 in high ATP concentration. These results could provide new ideas of the mechanism of BLM resolving G4. Our modified magnetic tweezer shows its capacity in G4 single molecule study, and it could be a useful tool in the future single molecule studies.

Actomyosin interaction at low ATP concentrations.

In vitro motility assay (IVMA) experiments were performed to analyze the movement of actin filaments sliding on a pavement of myosin molecules at different [ATP] and [ADP]. In standard experimental conditions at [ATP]=2mM, about 80% of the actin filaments move in unloaded conditions with a constant velocity. However, a fraction of at least 20% static actin filaments is always present. The accepted explanation is the occurrence of damaged "rigor"-like myosin heads that do not undergo the normal ATP-dependent cycling motion. However, in a series of IVMA experiments performed at different [ATP] we observed that the mobility of actin filaments increased with lowering [ATP]. We investigated the influence of [ATP] on the number of mobile actin filaments. IVMA experiments were performed at controlled nucleotide concentrations and the percentage of mobile filaments accurately determined by specific operator-guided software. The value of ΔG ATP involved was determined. Results showed that the number of mobile actin filaments sliding on type 2B heavy meromyosin isoform (2B HMM) increased at very low [ATP] accompanied by less negative ΔG ATP values. Similar results were obtained by increasing [ADP]. Performing experiments at the same [ATP] with different myosin types, we found a higher number of mobile actin filaments on slow type 1 HMM with respect to type 2B HMM while the highest number of mobile actin filaments was found on single-head myosin (S1 fraction). We also found that [ATP] did not influence the percentage of mobile actin filaments sliding on S1. Our results reveal novel aspects of actomyosin interaction.

Novel translocation intermediate allows re-evaluation of roles of ATP, proton motive force and SecG at the late stage of preprotein translocation.

Presecretory proteins such as pOmpA are translocated across the inner membrane of Escherichia coli by Sec translocase powered by ATP and proton motive force (PMF). Translocation activity has been determined by protease protection assaying invitro. We identified a new translocation intermediate at a late stage, which was protected by proteinase K (PK), but became PK sensitive upon urea extraction. At a late stage of pOmpA translocation driven by PMF in the presence of a nonhydrolyzable ATP analogue, the PK-protected materials arose, but were pulled back upon urea extraction, indicating that completion of translocation requires ATP hydrolysis. When inverted membrane vesicles prepared from secG-null strain (ΔSecG IMV) were used in the absence of PMF, the translocation intermediate was accumulated. When the ATP concentration was low in the absence of PMF, the translocation intermediate was also accumulated. Imposition of PMF in the presence of a low ATP concentration caused recovery of pOmpA translocation and resistance to urea extraction for SecG+ IMV, but not for ΔSecG IMV. Thus, analysis of the late translocation intermediate showed that two of three constituents, physiological concentration of ATP, PMF and SecG, are required for the catalytic cycle of preprotein translocation, that is, completion and subsequent initiation of translocation.

Andrographolide-loaded polymerized phenylboronic acid nanoconstruct for stimuli-responsive chemotherapy

Along with the successful discovery of paclitaxel as an anticancer drug, natural products have drawn great attention in drug discovery. Recently, andrographolide (AND) from Andrographis paniculata was reported to provide several benefits, including an anticancer effect. However, the extremely low solubility of the compound in an aqueous medium was an obstacle to overcome for the systemic administration and clinical application of AND. Based on our previous report, we formulated a water-soluble nanoconstruct by forming a boronic ester between the cis-1,3-diol of AND with hydrophilically polymerized phenylboronic acid (pPBA). The release of loaded AND was controlled by intracellular conditions, specifically, by low pH and high ATP concentrations, due to the pH- and diol-dependent affinity of the boronic ester. Because of the intrinsic property of the PBA moiety, the pPBA-AND nanoconstruct exhibited an excellent tumor targeting ability both in vitro and in vivo. Finally, a significant inhibition of tumor growth was observed in vivo. Taken together, our strategy, which is based on the formulation of a soluble nanoconstruct using hydrophilically polymerized PBA and a cis-diol, is plausible and provides a delivery system for a wide variety of chemotherapeutics. This strategy has applications not only in cancer therapy but also broader fields such as anti-inflammation or immunotherapy.

Abstract 4575: Altered catalytic properties of a subset of Met cytoplasmic domain variants occurring in renal cell carcinoma

Abstract TCGA provisional data of papillary renal cell carcinoma (PRCC) kidney cancer, a malignant hereditary cancer syndrome, reveals that 23% of patients (n = 161) have MET gene amplification and/or mRNA up-regulation (2-fold or greater; RNA-Seq V2) and 10% of patients possess MET mutations. The Met tyrosine kinase is the cell surface receptor for hepatocyte growth factor (HGF). HGF stimulation induces Met protein autophosphorylation and consequently enhances epithelial cell motility, morphogenesis, and proliferation. Chronic and/or overstimulation of Met contributes to tumor growth and metastasis. We determined Km and Vmax for ATP binding to full-length Met protein isolated from the PRCC derived cell line ACHN, which possesses the MET mutation T992I (numbered per SWISSPROT accession P08581) in the juxtamembrane domain, and the clear cell RCC derived cell line Caki-1, which possesses the MET V1220I mutation in the kinase domain. Catalytic constants were determined using two-site immunoassays for total Met and phospho-Met. Both variants were associated with increased Vmax relative to wild type Met protein isolated from B5/589 breast epithelial cells. The observed increased catalytic activity of receptor variants may confer a higher overall rate of signaling at lower ATP concentrations, potentially contributing to enhanced tumor cell motility and proliferation in both papillary and clear cell RCC. Citation Format: Dinuka De Silva, Young Lee, Arpita Roy, Donald P. Bottaro. Altered catalytic properties of a subset of Met cytoplasmic domain variants occurring in renal cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4575.

Modeling Interactions among Individual P2 Receptors to Explain Complex Response Patterns over a Wide Range of ATP Concentrations.

Extracellular ATP acts on the P2X family of ligand-gated ion channels and several members of the P2Y family of G protein-coupled receptors to mediate intercellular communication among many cell types including bone-forming osteoblasts. It is known that multiple P2 receptors are expressed on osteoblasts (P2X2,5,6,7 and P2Y1,2,4,6). In the current study, we investigated complex interactions within the P2 receptor network using mathematical modeling. To characterize individual P2 receptors, we extracted data from published studies of overexpressed human and rodent (rat and mouse) receptors and fit their dependencies on ATP concentration using the Hill equation. Next, we examined responses induced by an ensemble of endogenously expressed P2 receptors. Murine osteoblastic cells (MC3T3-E1 cells) were loaded with fluo-4 and stimulated with varying concentrations of extracellular ATP. Elevations in the concentration of cytosolic free calcium ([Ca2+]i) were monitored by confocal microscopy. Dependence of the calcium response on ATP concentration exhibited a complex pattern that was not explained by the simple addition of individual receptor responses. Fitting the experimental data with a combination of Hill equations from individual receptors revealed that P2Y1 and P2X7 mediated the rise in [Ca2+]i at very low and high ATP concentrations, respectively. Interestingly, to describe responses at intermediate ATP concentrations, we had to assume that a receptor with a K1∕2 in that range (e.g. P2Y4 or P2X5) exerts an inhibitory effect. This study provides new insights into the interactions among individual P2 receptors in producing an ensemble response to extracellular ATP.

Transcription Start Site Scanning and the Requirement for ATP during Transcription Initiation by RNA Polymerase II

Saccharomyces cerevisiae RNA polymerase (Pol) II locates transcription start sites (TSS) at TATA-containing promoters by scanning sequences downstream from the site of preinitiation complex formation, a process that involves the translocation of downstream promoter DNA toward Pol II. To investigate a potential role of yeast Pol II transcription in TSS scanning, HIS4 promoter derivatives were generated that limited transcripts in the 30-bp scanned region to two nucleotides in length. Although we found that TSS scanning does not require RNA synthesis, our results revealed that transcription in the purified yeast basal system is largely ATP-independent despite a requirement for the TFIIH DNA translocase subunit Ssl2. This result is rationalized by our finding that, although they are poorer substrates, UTP and GTP can also be utilized by Ssl2. ATPγS is a strong inhibitor of rNTP-fueled translocation, and high concentrations of ATPγS make transcription completely dependent on added dATP. Limiting Pol II function with low ATP concentrations shifted the TSS position downstream. Combined with prior work, our results show that Pol II transcription plays an important role in TSS selection but is not required for the scanning reaction.

Role of lutoid membrane transport and protein synthesis in the regeneration mechanism of latex in different Hevea clones

Natural rubber (cis-1,4 polyisoprene) is synthesised in the milky cytoplasm, the latex, of specialized cells called laticifers in the bark tissues of the rubber tree (Hevea brasiliensis). Regeneration mechanism of latex after each tapping (controlled wounding of the bark) was studied in relation to lutoid membrane enzymes and protein synthesis in twelve rubber clones with varying yield potentials during the peak rubber yielding season. High activity of membrane enzymes and better availability of biochemical energy [ATP] were observed in clones viz; RRII 105, RRIM 600, PB 260, RRII 422 and RRII 430. The highest protein biosynthetic capacity was noticed in clone PB 260 and RRIM 600. However, high ATP content, increased invertase activity and protein biosynthesis were observed in the medium yielding clone GT1 compared to clones with low rubber yield potential. Very low sugar content and increased invertase activity in the latex of clone PB 260 indicated intense latex metabolism with high protein turnover that implies fast recouping of the cellular metabolites lost during latex harvesting. Clone PB 217 was characterized by very high sucrose and low ATP concentration and ATPase activity in latex indicating slow metabolism and hence be suitable for inducing latex metabolism using ethylene stimulant. Low rubber yielding clones such as RRII 33 and RRII 38 were consistently recorded a high sucrose content but very low activity of membrane enzymes, reduced ATP concentration and low protein biosynthesis in latex. Among the recently released modern clones (RRII 400 series), latex regeneration capacity was higher in RRII 422 and RRII 430. The significance of lutoid membrane transport and protein synthesis is discussed in relation to general latex metabolism of these rubber clones. The outcome of this study would be helpful to design suitable latex harvesting systems and yield stimulation methods for optimizing latex production in each clone based on metabolic profiling.

P2X7 nucleotide receptor signaling potentiates the Wnt/β-catenin pathway in cells of the osteoblast lineage.

The P2X7 and Wnt/β-catenin signaling pathways regulate osteoblast differentiation and are critical for the anabolic responses of bone to mechanical loading. However, whether these pathways interact to control osteoblast activity is unknown. The purpose of this study was to investigate the effects of P2X7 activation on Wnt/β-catenin signaling in osteoblasts. Using MC3T3-E1 cells, we found that combined treatment with Wnt3a and the P2X7 agonist 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) elicited more sustained β-catenin nuclear localization than that induced by Wnt3a alone. Wnt3a-induced increases in β-catenin transcriptional activity were also potentiated by treatment with BzATP. Consistent with involvement of P2X7, a high ATP concentration (1mM) potentiated Wnt3a-induced β-catenin transcriptional activity, whereas a low concentration (10μM) of ATP, adenosine 5'-diphosphate (ADP), or uridine 5'-triphosphate (UTP) failed to elicit a response. The potentiation of β-catenin transcriptional activity elicited by BzATP was also inhibited by two distinct P2X7 antagonists: A 438079 and A 740003. Furthermore, responses to Wnt3a in calvarial cells isolated from P2rx7 knockout mice were significantly less than in cells from wild-type controls. In MC3T3-E1 cells, BzATP increased inhibitory phosphorylation of glycogen synthase kinase 3β (GSK3β), a process that was blocked by A 438079 and diminished by inhibition of protein kinase C. Thus, P2X7 signaling may potentiate the canonical Wnt pathway through GSK3β inhibition. Taken together, we show that P2X7 activation prolongs and potentiates Wnt/β-catenin signaling. Consequently, cross-talk between P2X7 and Wnt/β-catenin pathways may modulate osteoblast activity in response to mechanical loading.

An investigation into the role of ATP in the mammalian pre-mRNA 3' cleavage reaction.

RNA Polymerase II transcribes beyond what later becomes the 3' end of a mature messenger RNA (mRNA). The formation of most mRNA 3' ends results from pre-mRNA cleavage followed by polyadenylation. Invitro studies have shown that low concentrations of ATP stimulate the 3' cleavage reaction while high concentrations inhibit it, but the origin of these ATP effects is unknown. ATP might enable a cleavage factor kinase or activate a cleavage factor directly. To distinguish between these possibilities, we tested several ATP structural analogs in a pre-mRNA 3' cleavage reaction reconstituted from DEAE-fractionated cleavage factors. We found that adenosine 5'-(β,γ-methylene)triphosphate (AMP-PCP) is an effective invitro 3' cleavage inhibitor with an IC50 of ∼300μM, but that most other ATP analogs, including adenosine 5'-(β,γ-imido)triphosphate, which cannot serve as a protein kinase substrate, promoted 3' cleavage but less efficiently than ATP. In combination with previous literature data, our results do not support ATP stimulation of 3' cleavage through cleavage factor phosphorylation invitro. Instead, the more likely mechanism is that ATP stimulates cleavage factor activity through direct cleavage factor binding. The mammalian 3' cleavage factors known to bind ATP include the cleavage factor II (CF IIm) Clp1 subunit, the CF Im25 subunit and poly(A) polymerase alpha (PAP). The yeast homolog of the CF IIm complex also binds ATP through yClp1. To investigate the mammalian complex, we used a cell-line expressing FLAG-tagged Clp1 to co-immunoprecipitate Pcf11 as a function of ATP concentration. FLAG-Clp1 co-precipitated Pcf11 with or without ATP and the complex was not affected by AMP-PCP. Diadenosine tetraphosphate (Ap4A), an ATP analog that binds the Nudix domain of the CF Im25 subunit with higher affinity than ATP, neither stimulated 3' cleavage in place of ATP nor antagonized ATP-stimulated 3' cleavage. The ATP-binding site of PAP was disrupted by site directed mutagenesis but a reconstituted 3' cleavage reaction containing a mutant PAP unable to bind ATP nevertheless underwent ATP-stimulated 3' cleavage. Fluctuating ATP levels might contribute to the regulation of pre-mRNA 3' cleavage, but the three subunits investigated here do not appear to be responsible for the ATP-stimulation of pre-mRNA cleavage.

Essential Involvement of Purinergic Signaling in Calcium Handling in the Podocytes of Type 2 Diabetic Rats

Purinergic signaling (initiated via interaction of ATP with P2 receptors) is one of the major triggers of intracellular calcium influx in the epithelial cells of the glomeruli ‐ podocytes. Excessive elevation of intracellular calcium concentration in these cells results in their loss, subsequent damage of the glomeruli filtration barrier and, eventually, proteinuria. Growing evidence suggests that the activation of extracellular ATP signaling may play a crucial role in the development of diabetic nephropathy. The goal of this study was to characterize purinergic signaling in the podocytes of Type 2 Diabetic (T2D) rats. Here we used 12 week old and aged (40–60 weeks old) Wistar (control), Goto‐Kakizaki (GK) and Type 2 Diabetic Nephropathy (T2DN) rats. The GK rat is a non‐obese Wistar substrain which develops T2D early in life. T2DN rat (created by an introgression of the mitochondria and some passenger loci from the Fawn Hooded Hypertensive (FHH) rat into the background of GK rat) is a model of T2D accompanied with renal histologic abnormalities characteristic for diabetic nephropathy (DN). Using real‐time confocal microscopy we measured acute effects of ATP on intracellular calcium dynamics in the podocytes of glomeruli freshly isolated from these rat strains. We have shown that basal intracellular calcium concentration was within normal range in Wistar and GK podocytes (150.1 ± 10.8 and 174.7 ± 29.7 nM, respectively), whereas in T2DN rats it was pathologically elevated up to 232.9 ± 20.1 nM. Effect of ATP in Wistar rat podocytes (in the range of 1 to 200 μM) was dose dependent and similar to what we previously reported for Sprague Dawley rats. However, ATP‐triggered calcium influx in GK rats was substantially enhanced with dose‐response curve shifted towards stronger activation of calcium influx with lower concentrations of ATP. Importantly, the response to ATP was further increased in T2DN compared to GK rat podocytes, and was found to be dose‐independent within the same ATP concentration range. The same phenomenon was observed in aged (approximately 12 month old) GK and T2DN animals. Furthermore, experiments in calcium‐free solutions and studies using alpha, beta‐methylene‐ATP revealed a major role of extracellular calcium influx as distinct from intracellular depot depletion, as well as a likely involvement of the ionotropic P2X receptors, or calcium channels activated by P2Y‐evoked metabotropic GPCR cascades. Western blotting revealed a significantly higher expression of P2X7 receptors in the cortex of T2DN and GK strains compared to Wistar rats, and a decrease in P2Y1 expression. Thus, our data indicate that in T2DN podocytes ATP‐triggered calcium influx is enhanced compared to control animals with even low concentrations of ATP causing activation of calcium flux and augmented intracellular calcium concentration, correspondingly. This mechanism might be an important determinant of podocyte injury resulting from pathological remodeling from metabotropic P2Y to ionotropic P2X receptors occurring in DN.Support or Funding InformationSupported by ASN Ben J. Lipps fellowship, ADA 1‐15‐BS‐172, NIH DK105160 and HL122662.

Independent Control of the Static and Dynamic Components of the Chlamydomonas Flagellar Beat

When the green alga Chlamydomonas reinhardtii swims, it uses the breaststroke beat of its two flagella to pull itself forward [1]. The flagellar waveform can be decomposed into a static component, corresponding to an asymmetric time-averaged shape, and a dynamic component, corresponding to the time-varying wave [2]. Extreme lightening conditions photoshock the cell, converting the breaststroke beat into a symmetric sperm-like beat, which causes a reversal of the direction of swimming [3]. Waveform conversion is achieved by a reduction in magnitudeofthe static component, whereas the dynamic component remains unchanged [2]. The coupling between static and dynamic components, however, is poorly understood, and it is not known whether the static component requires the dynamic component or whether it can exist independently. We used isolated and reactivated axonemes [4] to investigate the relation between the two beat components. We discovered that, when reactivated in the presence of low ATP concentrations, axonemes displayed the static beat component in absence of the dynamic component. Furthermore, we found that the amplitudes of the two components depend on ATP in qualitatively different ways. These results show that the decomposition into static and dynamic components is not just a mathematical concept but that the two components can independently control different aspects of cell motility: the static component controls swimming direction, whereas the dynamic component provides propulsion.

Spectroscopic Studies of the EutT Adenosyltransferase from Salmonella enterica: Mechanism of Four-Coordinate Co(II)Cbl Formation.

EutT from Salmonella enterica is a member of a class of enzymes termed ATP:Co(I)rrinoid adenosyltransferases (ACATs), implicated in the biosynthesis of adenosylcobalamin (AdoCbl). In the presence of cosubstrate ATP, ACATs raise the Co(II)/Co(I) reduction potential of their cob(II)alamin [Co(II)Cbl] substrate by >250 mV via the formation of a unique four-coordinate (4c) Co(II)Cbl species, thereby facilitating the formation of a "supernucleophilic" cob(I)alamin intermediate required for the formation of the AdoCbl product. Previous kinetic studies of EutT revealed the importance of a HX11CCX2C(83) motif for catalytic activity and have led to the proposal that residues in this motif serve as the binding site for a divalent transition metal cofactor [e.g., Fe(II) or Zn(II)]. This motif is absent in other ACAT families, suggesting that EutT employs a distinct mechanism for AdoCbl formation. To assess how metal ion binding to the HX11CCX2C(83) motif affects the relative yield of 4c Co(II)Cbl generated in the EutT active site, we have characterized several enzyme variants by using electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopies. Our results indicate that Fe(II) or Zn(II) binding to the HX11CCX2C(83) motif of EutT is required for promoting the formation of 4c Co(II)Cbl. Intriguingly, our spectroscopic data also reveal the presence of an equilibrium between five-coordinate "base-on" and "base-off" Co(II)Cbl species bound to the EutT active site at low ATP concentrations, which shifts in favor of "base-off" Co(II)Cbl in the presence of excess ATP, suggesting that the base-off species serves as a precursor to 4c Co(II)Cbl.

Unique PFK regulatory property from some mosquito vectors of disease, and from Drosophila melanogaster.

BackgroundArthropod-borne diseases are some of the most rapidly spreading diseases. Reducing the vector population is currently the only effective way to reduce case numbers. Central metabolic pathways are potential targets to control vector populations, but have not been well explored to this aim. The information available on energy metabolism, as a way to control lifespan and dispersion through flight of dipteran vectors, is inadequate.MethodsPhosphofructokinase (PFK) activity was measured in the presence of both of its substrates, fructose-6-phosphate (F6P) and ATP, as well as some allosteric effectors: Fructose- 2,6 – bisphosphate (F2, 6BP), citrate and AMP. Aedes aegypti phosphofructokinase sequence (AaPFK) was aligned with many other insects and also vertebrate sequences. A 3D AaPFK model was produced and docking experiments were performed with AMP and citrate.ResultsThe kinetic parameters of AaPFK were determined for both substrates: F6P (V = 4.47 ± 0.15 μmol of F1, 6BP/min, K0.5 = 1.48 ± 0.22 mM) and ATP (V = 4.73 ± 0.57 μmol of F1, 6BP/min, K0.5 = 0.43 ± 0.10 mM). F2,6P was a powerful activator of AaPFK, even at low ATP concentrations. AaPFK inhibition by ATP was not enhanced by citrate, consistent with observations in other insects. After examining the sequence alignment of insect and non-insect PFKs, the hypothesis is that a modification of the citrate binding site is responsible for this unique behavior. AMP, a well-known positive effector of PFK, was not capable of reverting ATP inhibition. Aedes, Anopheles and Culex are dengue, malaria and filariasis vectors, respectively, and are shown to have this distinct characteristic in phosphofructokinase control. The alignment of several insect PFKs suggested a difference in the AMP binding site and a significant change in local charges, which introduces a highly negative charge in this part of the protein, making the binding of AMP unlikely. This hypothesis was supported by 3D modeling of PFK with AMP docking, which suggested that the AMP molecule binds in a reverse orientation due to the electrostatic environment. The present findings imply a potential new way to control PFK activity and are a unique feature of these Diptera.ConclusionsThe present findings provide the first molecular explanation for citrate insensitivity in insect PFKs, as well as demonstrating for the first time AMP insensitivity in dipterans. It also identified a potential target for novel insecticides for the control of arthropod-borne diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1391-y) contains supplementary material, which is available to authorized users.

Inferring Growth Control Mechanisms in Growing Multi-cellular Spheroids of NSCLC Cells from Spatial-Temporal Image Data

We develop a quantitative single cell-based mathematical model for multi-cellular tumor spheroids (MCTS) of SK-MES-1 cells, a non-small cell lung cancer (NSCLC) cell line, growing under various nutrient conditions: we confront the simulations performed with this model with data on the growth kinetics and spatial labeling patterns for cell proliferation, extracellular matrix (ECM), cell distribution and cell death. We start with a simple model capturing part of the experimental observations. We then show, by performing a sensitivity analysis at each development stage of the model that its complexity needs to be stepwise increased to account for further experimental growth conditions. We thus ultimately arrive at a model that mimics the MCTS growth under multiple conditions to a great extent. Interestingly, the final model, is a minimal model capable of explaining all data simultaneously in the sense, that the number of mechanisms it contains is sufficient to explain the data and missing out any of its mechanisms did not permit fit between all data and the model within physiological parameter ranges. Nevertheless, compared to earlier models it is quite complex i.e., it includes a wide range of mechanisms discussed in biological literature. In this model, the cells lacking oxygen switch from aerobe to anaerobe glycolysis and produce lactate. Too high concentrations of lactate or too low concentrations of ATP promote cell death. Only if the extracellular matrix density overcomes a certain threshold, cells are able to enter the cell cycle. Dying cells produce a diffusive growth inhibitor. Missing out the spatial information would not permit to infer the mechanisms at work. Our findings suggest that this iterative data integration together with intermediate model sensitivity analysis at each model development stage, provide a promising strategy to infer predictive yet minimal (in the above sense) quantitative models of tumor growth, as prospectively of other tissue organization processes. Importantly, calibrating the model with two nutriment-rich growth conditions, the outcome for two nutriment-poor growth conditions could be predicted. As the final model is however quite complex, incorporating many mechanisms, space, time, and stochastic processes, parameter identification is a challenge. This calls for more efficient strategies of imaging and image analysis, as well as of parameter identification in stochastic agent-based simulations.