ATPase activities in partially purified membranes of Acetabularia

Partly purified membranes (with plasmalemma material) of Acetabularia mediterranea were studied with respect to ATPase activity in alkali- and Ca⁺⁺-free media and its sensitivity to pH (5 – 9), oligomycin (200 ?g/mg protein), 100 ?M N-N′-dicyclohexylcarbodiimide (DCCD), and 50 ?M vanadate. Besides activities which may originate from mitochondrial H⁺ ATPase (oligomycin-sensitive, alkaline pH optimum) and tonoplast H⁺ ATPase (DCCD-sensitive, pH optimum 7.5), there is ATPase activity with a pH optimum around pH 6.5, sensitive to vanadate and insensitive to DCCD. These results strongly suggest that the electrogenic Cl^? pump in the plasmalemma of Acetabularia is an ATPase. Effects of Mg⁺⁺, Mg-ATP, ADP, GTP, UTP, CTP and HCO₃ ^? versus Cl^? on this ATPase activity are described.     

Binding of mitochondrial ATPase from ox heart to its naturally occurring inhibitor protein: Localization by antibody binding

The naturally occurring ATPase inhibitor protein from ox heart mitochondria was cross-linked to its binding site on the mitochondrial ATPase using 1-ethyl-3-(dimethylamino)propyl carbodiimide. The cross-linked product, when transferred electrophoretically to a nitrocellulose sheet, reacted with antibodies directed against the inhibitor protein and the β-subunit of the ATPase. It was concluded that the binding site for the inhibitor protein lies on the β-subunit.     

Regulation of sodium in the shore crabCarcinus maenas,adapted to environments of constant and changing salinities

The activity of Na-K-ATPase was determined in the posterior gills of the shore crabCarcinus maenas during a period following transfer from 35 to 10 ‰ salinity and vice versa at 15 °C. After transfer from high to low salinity, Na-K-ATPase activity increased from 3.2 to 7.0 μmoles P_i mg protein^?1 h^?1 within a period of 2 to 3 weeks. Transfer of crabs from low to high salinity resulted in reduction of activity from 7.4 to 4.5 μmoles P_i mg protein^?1 h^?1 within about the same period. The relatively slow response following salinity change indicates that the amounts of Na-K-ATPase in the gills may play a role in hyperionic Na regulation in relatively constant brackish-water environments. Instant responses to salinity result from activation and inhibition of Na-K-ATPase activity by Na. Gill Na-K-ATPase is activated by the Na concentration of the incubation medium to attain a steep maximum at about 75 mM Na, which corresponds to the lowest environmental Na levels tolerated byC. maenas equivalent to a salinity of ca 6 ‰. Activity greatly decreased towards higher Na levels, equivalent to the salinity of normal sea water, at which hyperregulation no longer occurs. Selective addition of either Na or Cl to brackish water of 9 ‰ S resulted in effective hyperregulation of the non-increased ion, and passive distribution between medium and blood of the increased ion. These data indicate that under appropriate conditions the normally coupled transport of Na and Cl may be uncoupled and take place independently of each other.     

Phloem unloading in aerial roots of Monstera deliciosa

Plants of Monstera deliciosa Liebm. pruned to exemplars with one leaf and one aerial root were labeled with 7.4 MBq ¹⁴CO₂ over the leaf blade. Microautoradiographs of soluble and insoluble radioactivity were prepared from three different regions of the aerial root. In addition, histochemical localization of ATPase was carried out on similar aerial roots. Vigorously growing aerial roots grew as fast as 26 mm d^-1, and zones of differentiation extended more than 10 cm from the root tip. In the region 2–3 cm from the root tip, in which only protoelements of the vascular tissue were differentiated, ¹⁴C-label was restricted to the protophloem. The activity of ATPase was recognized in many different cellular organelles of the meristematic phloem parenchyma. In the region 5–6 cm from the root tip, in which the first metaelements differentiated, all parenchyma cells of the central cylinder and many cortical cells showed ¹⁴C-label, in addition to the densely labeled protophloem. Differentiating vessels were heavily labeled at sites where secondary walls were formed. In this region of the root, ATPase activity was concentrated on the plasmalemma and cortical cytoplasma of the sieve tubes, and on the tonoplast of the phloem parenchyma cells. In contrast, the strands of internal metaphloem with giant sieve tubes, which are scattered among the metaxylem, were neither labeled nor did they show ATPase activity. In the zone 19–20 cm from the root tip, regions of cell differentiation in the sclerenchymatic mantle of the inner cortex, the late-formed metaxylem vessels and some strands of the internal metaphloem could be identified by dense ¹⁴C-label. Low ATPase activity was found in the plasmalemma of practically all living cells. In this nearly mature region, a strong peroxidase activity was observed in the radial walls of the endodermis. The results indicate that phloem unloading was strongest at sites of root differentiation, where ATPase activity was concentrated in the plasmalemma of sieve tubes and the tonoplast of phloem parenchyma.     

Interaction of a coupling factor from Rhodospirillum rubrum with coupling factor deficient chromatophores

A coupling factor necessary for the photophosphorylation and Mg²⁺-ATPase activities of Rhodospirillum rubrum chromatophores has been separated from these particles. Although the redox potential of coupling factor deficient chromatophores is slightly more oxidized than of the control, the addition of the coupling factor for reconstitution does not alter the redox potential. Phenazine methosulfate cannot restore or significantly enhance the photophosphorylation activities of uncoupled or reconstituted chromatophores compared to the control. The coupling factor can bind to coupling factor deficient membranes without addition of magnesium ions and thus restore the photophosphorylation and Mg²⁺-ATPase activities of these vesicles. The Ca²⁺-ATPase in the coupling factor preparation shows binding characteristics similar to those of the coupling factor.     

Characteristics of (Na++K+)-ATPase inBoergesenia forbesii

(Na⁺+K⁺)-ATPase proved to be present in the vegetative thalli ofBoergesenia forbesii (Harvey) Feldmann. The ATPase was extracted with Triton X-100 and partially purified by Sephadex G-150 gel filtration. The enzyme was activated with Mg²⁺ and further stimulated by the addition of K⁺ and Na⁺. It was observed thatp-chloromercuribenzoate (PCMB),N-ethylmaleimide (NEM), iodoacetoamide, copper sulfate, zinc sulfate, lead nitrate and cadmium chloride inhibited the enzyme activity, but ouabain was ineffective, andN,N′-dicyclohexylcarbodiimide (DCCD) did not apparently inhibit the activity, but rather promoted it slightly. The ATPase activity was also shown in the isolated cell wall ofBoergesenia thalli, and the enzyme activity was detected in the wall itself by using electron microscopic methods.     

Studies on the regulatory complex of rabbit skeletal muscle: Contributions of troponin subunits and tropomyosin in the presence and absence of Mg2+ to the acto-S1 ATPase activity

The regulatory roles of the components of the troponin-tropomyosin complex in the presence and absence of Mg²⁺ on the acto-S1 ATPase have been examined. The effect of free Mg²⁺ on the inhibition of the acto-S1 ATPase by rabbit skeletal troponin (Tn) was studied at S1 to actin ratios ranging from 0.17:1 to 2.5:1. These studies were performed using two Mg²⁺ concentrations: 2.5 mM Mg²⁺-2.5 mM ATP, conditions considered to have low free Mg²⁺; and 5.0 mM Mg²⁺-2.5 mM ATP, conditions providing a high free Mg²⁺ concentration of ～2.5 mM. In the presence of high free Mg²⁺ (2.5 mM ATP-5.0 mM MgCl₂) the Tn inhibition of acto-S1-TM ATPase increased by approximately 40–50% over a range of S1 to actin ratios of 0.17:1 to 2.5:1. The effect of free Mg²⁺ on increasing quantities of Tn in the absence or presence of tropomyosin was studied independently at two S1 to actin ratios (1:1 and 2:1). In the absence of TM, at 5 mM Mg²⁺ there is an additional 38% (1:1 S1 to actin) or 37% (2:1) decrease in the ATPase activity by Tn compared to 2.5 mM Mg²⁺. Similarly, in the presence of TM and Tn, Mg²⁺ exerts its effect at both S1 to actin ratios. Significantly, the inhibition by the IT complex in the presence of TM is unaffected by free Mg²⁺. Furthermore, ultracentrifugation binding studies using¹⁴C-iodoacetamide-labeled Tn and TM established that the Tn-TM regulatory complex was firmly bound to F-actin at both Mg²⁺ concentrations, indicating that faciliation of binding to F-actin by Mg²⁺ is not responsible for the increased inhibition. Hence, it is concluded from the data that Mg²⁺ binding and by analogy Ca²⁺ binding to the Ca²⁺-Mg²⁺ sites of TnC promotes muscle relaxation by inducing inhibition of the actomyosin ATPase, whereas Ca²⁺ binding to the Ca²⁺-specific sites promotes contraction by potentiating the ATPase. The inhibition of the acto-S1-TM ATPase by TnT has also been further examined. The data indicate that TnT exerts the same level of inhibition upon the ATPase as TnI or Tn. The inhibitory activity requires TM, and occurs to the same extent under conditions where TM alone would have either a potentiating (2:1 S1 to actin) or an inhibitory (1:1 S1 to actin) effect upon the ATPase. In the presence of TM the IT complex is a more effective inhibitor than either TnI, TnT, or Tn. The inhibitory activity of the IT complex is partially released by TnC in the absence of Ca²⁺. These observations, in conjunction with those by Chong, Asselbergs, and Hodges, which showed that the inhibition by TnT is partially released by TnC plus Ca²⁺, indicate that the role of TnT involves more than anchoring Tn to the thin filament.     

Evidence for an electrogenic adenosine-triphosphatase in Hevea tonoplast vesicles

The function of the Mg-dependent ATPase of Hevea tonoplast in active proton transport was investigated by using a purified tonoplast fraction containing tightly sealed vesicles. In the used experimental conditions, the uptake of [¹⁴C]triphenylmethyl-phosphonium ion ([¹⁴C]TPMP⁺) and [³H]tetraphenyl-phosphonium ion ([³H]TPP⁺) by the vesicles indicated a transmembrane potential difference, negative inside. In parallel, the uptake of [¹⁴C]methylamine into the vesicles monitored a transmembrane pH gradient, interior acid. The addition of 5 mM Mg-ATP markedly depolarized the membrane and increased the magnitude of trnasmembrane pH gradient. These ATP-driven events were substrate specific for Mg-ATP. They were strongly inhibited by ATPase inhibitors such as N, N′-dicyclohexylcar-bodiimide. They were completely eliminated by proton conductors such as carbonylcyanide-p-trifluoromethoxy-phenylhydrazone and 5-chloro-3-tert-butyl-2′-chloro-4-nitro-salicylanilide. They depended on the pH of the medium, the maximum being reached at about pH 7.0. These data provide in vitro evidence that the Mg-ATPase localized at tonoplast level is an electrogenic pump. They are consistent with the hypothesis that an electrogenic H⁺ pump is catalyzed by the tonoplast ATPase of higher plants.     

Apparent Reduction of ADAM10 in Scrapie-Infected Cultured Cells and in the Brains of Scrapie-Infected Rodents

It has been described that A disintegrin and metalloproteinase (ADAM10) may involve in the physiopathology of prion diseases, but the direct molecular basis still remains unsolved. In this study, we confirmed that ADAM10 was able to cleave recombinant human prion protein in vitro. Using immunoprecipitation tests (IP) and immunofluorescent assays (IFA), reliable molecular interaction between the native cellular form of PrP (PrP^C) and ADAM10 was observed not only in various cultured neuronal cell lines but also in brain homogenates of healthy hamsters and mice. Only mature ADAM10 (after removal of its prodomain) molecules showed the binding activity with the native PrP^C. Remarkably more prion protein (PrP)-ADAM10 complexes were detected in the membrane fraction of cultured cells. In the scrapie-infected SMB cell model, the endogenous ADAM10 levels, especially the mature ADAM10, were significantly decreased in the fraction of cell membrane. IP and IFA tests of prion-infected SMB-S15 cells confirmed no detectable PrP-ADAM10 complex in the cellular lysates and PrP-ADAM10 co-localization on the cell surface. Furthermore, we demonstrated that the levels of ADAM10 in the brain homogenates of scrapie agent 263K-infected hamsters and agent ME7-infected mice were also almost diminished at the terminal stage, showing time-dependent decreases during the incubation period. Our data here provide the solid molecular basis for the endoproteolysis of ADAM10 on PrP molecules and interaction between ADAM10 and PrP^C. Obvious loss of ADAM10 during prion infection in vitro and in vivo highlights that ADAM10 may play essential pathophysiological roles in prion replication and accumulation.     

Cadmium,mercury, and lead effects on gill tissue of freshwater crayfishProcambarus clarkii (girard)

Intermolt adult crayfishP. clarkii were used for this work. After acclimatation to laboratory conditions crayfish were exposed to sublethal concentrations of cadmium, mercury, and lead for 96 h. Gills of control and exposed crayfish were removed and ATPase activity and oxygen uptake rate were determined. Structural damage of gill filaments was also observed. Gill tissue respiration rates were measured for individual crayfish using a Gilson differential respirometer. Lead causes a decrease of gill oxygen uptake, but neither cadmium nor mercury seems to affect it at the concentrations employed. Although all metals studied alter gill filament structure, lead damage is the most apparent. In the same way, significant differences in gill ATPase activity owing to metal exposure were only observed in lead treated crayfish.     

Role of α-helical domains in functioning of ATP-dependent Lon protease of Escherichia coli

Homooligomeric ATP-dependent LonA proteases are bifunctional enzymes belonging to the superfamily of AAA⁺ proteins. Their subunits are formed by five successively connected domains, i.e., N-terminal (N), α-helical (HI(CC)), nucleotide-binding (NB), the second α-helical (H), and proteolytic (P) domains. The presence of the inserted HI(CC) domain determines the uniqueness of LonA proteases among the AAA⁺ proteins. The role of the α-helical domains in the LonA protease functioning was studied with an example of E. coli Lon protease (Ec-Lon). The properties of the intact Ec-Lon and its mutant forms, i.e., Lon-R164A and Lon-R542A bearing the substituted arginine residues at the similar positions in the HI(CC) and H domains, were compared. The H domain was shown to play a crucial role in ATP hydrolysis and enzyme binding to the target protein. The HI(CC) domain is not decisive for the manifestation of the catalytic properties of the enzyme. However, it affects the functioning of Lon ATPase and peptidase sites and is involved in maintaining enzyme stability. The participation of the HI(CC) domain in the formation of three-dimensional structures of LonA proteases and/or their complexes with DNA is suggested.     

Polymorphism of DNA Repair Gene xrcc1 and Lung Cancer Risk

The current large-scale meta-analysis was performed to reach a reliable conclusion on the association between X-ray repair cross-complementing 1 (xrcc1) rs1799782 and the development of lung cancer. Studies that investigated the association between rs1799782 and lung cancer risk were identified by searching PubMed. We calculated odds ratio (OR) with corresponding 95 % confidence interval (CI) for Trp/Trp vs Arg/Arg, Trp/Trp + Arg/Trp vs Arg/Arg, and Trp/Trp vs Arg/Trp + Arg/Arg contrast models. Combining all 25 studies, we yielded three summary ORs: 1.07 (95 % CI 0.92–1.23) for Trp/Trp vs Arg/Arg, 0.93 (95 % CI 0.87–1.00) for Trp/Trp + Arg/Trp vs Arg/Arg, and 1.08 (95 % CI 0.94–1.25) for Trp/Trp vs Arg/Trp + Arg/Arg, suggesting rs1799782 was not associated with overall risk of lung cancer. Strikingly, a significantly deceased risk was found among Caucasian populations (Trp/Trp + Arg/Trp vs Arg/Arg, OR = 0.86, 95 % CI 0.76–0.97). This study confirms that xrcc1 rs1799782 may lower the risk of lung cancer among Caucasians.     

Modulation,Bioinformatic Screening,and Assessment of Small Molecular Peptides Targeting the Vascular Endothelial Growth Factor Receptor

Vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) are important factors in tumor growth and metastasis. Molecular probes or drugs designed to target VEGF/VEGFR interactions are crucial in tumor molecular imaging and targeted therapy. Bioinformatic methods enable molecular design based on the structure of bio-macromolecules and their interactions. This study was aimed to identify tumor-targeting small-molecule peptides with high affinity for VEGFR using bioinformatics screening. The VEGFR extracellular immunoglobulin-like modules Ig1–Ig3 were used as the target to systematically alter the primary peptide sequence of VEGF_125–136. Molecular docking and surface functional group interaction methods were combined in an in silico screen for polypeptides, which in theory, would have higher affinities for VEGFR. In vitro receptor competition binding assays were used to assess the affinity of the putative VEGFR-binding polypeptides. Rhodamine-conjugated peptides were used to label and visualize peptide-binding sites on A549 cells. Using bioinformatic screening, we identified 20 polypeptides with potentially higher affinity for VEGFR. The polypeptides were capable of inhibiting the binding of ¹²⁵I-VEGF to VEGFR in a dose-dependent manner. The IC₅₀ values of QKRKRKKSRKKH and RKRKRKKSRYIVLS (80 and 185 nmol/L, respectively) were significantly lower than that of VEGF_125–136 (464 nmol/L); thus, the affinity of these peptides for VEGFR was 6- and 2.5-fold higher, respectively, than that of VEGF_125–136. Rhodamine labeling of A549 cells revealed peptide binding mainly on the plasma membrane and in the cytoplasm. Bioinformatic approaches hold promise for the development of molecular imaging probes. Using this approach, we designed two peptides that showed higher affinity toward VEGFR. These polypeptides may be used as molecular probes or drugs targeting VEGFR, which can be utilized in molecular imaging and targeted therapy of certain tumors.     

EGFR-AKT-mTOR activation mediates epiregulin-induced pleiotropic functions in cultured osteoblasts

Epidermal growth factor (EGF) receptor (EGFR) emerges as an essential molecule for the regulating of osteoblast cellular functions. In the current study, we explored the effect of epiregulin, a new EGFR ligand, on osteoblast functions in vitro, and studied the underlying mechanisms. We found that epiregulin-induced EGFR activation in both primary osteoblasts and osteoblast-like MC3T3-E1 cells. Meanwhile, epiregulin activated AKT-mammalian target of rapamycin (mTOR) and Erk-mitogen-activated protein kinase (MAPK) signalings in cultured osteoblasts, which were blocked by EGFR inhibitor AG1478 or monoclonal antibody against EGFR (anti-EGFR). Further, in primary and MC3T3-E1 osteoblasts, epiregulin promoted cell proliferation and increased alkaline phosphatase activity, while inhibiting dexamethasone (Dex)-induced cell death. Such effects by epiregulin were largely inhibited by AG1478 or anti-EGFR. Notably, AKT-mTOR inhibitors, but not Erk inhibitors, alleviated epiregulin-induced above pleiotropic functions in osteoblasts. Meanwhile, siRNA depletion of Sin1, a key component of mTOR complex 2 (mTORC2), also suppressed epiregulin-exerted effects in MC3T3-E1 cells. Together, these results suggest that epiregulin-induced pleiotropic functions in cultured osteoblasts are mediated through EGFR-AKT-mTOR signalings.     

Mechanism of bFGF-binding Peptide Reversing Adriamycin Resistance in Human Gastric Cancer Cells

Development of drug resistance is a challenging problem in cancer chemotherapy. It has been shown that basic fibroblast growth factor (bFGF) plays an important role in an epigenetic mechanism of drug resistance. We have isolated a bFGF binding peptide P7 with inhibitory activity against bFGF-induced proliferation of human gastric cancer cells by screening a phage display library. In this study, we found that P7 peptide also has efficacy of reversing bFGF-induced resistance to Adriamycin (ADM) in human gastric cancer cells. Further investigations with SGC-7901 cells revealed that inhibition of Akt activation triggered by bFGF, and reversal of bFGF-induced up-regulation of Bcl-2 and XIAP and down-regulation of Bax, contribute to P7 peptide counteracting the anti-apoptotic effect of bFGF, and further reversing bFGF-induced resistance to ADM. The results suggested that the bFGF-binding peptide may have therapeutic potential of drug resistance in gastric cancer.     

On the existence of two GABA pools associated with newly synthesized GABA and with newly taken up GABA in nerve terminals

[¹⁴C]Glutamic acid and [³H]GABA were injected into the lateral ventricle of mouse and then [¹⁴C]GABA and [³H]GABA in synaptosomes isolated from the animals were analysed. The [¹⁴C]GABA was interpreted to be newly synthesized GABA from [¹⁴C]glutamic acid while the [³H]GABA to be newly taken up GABA. We have obtained the following results: (1) when the animals were pretreated with aminooxyacetic acid and thus the GABA content in synaptosomes increased to about 2 times of the control level, only the [³H]GABA was enhanced to 3 times of the control level without any change of [¹⁴C]GABA, (2) the release of [¹⁴C]GABA from synaptosomes by high K⁺ depolarization was 1.5 times greater than that of [³H]GABA, (3) the releases of both [¹⁴C]GABA and [³H]GABA were increased in the presence of cold GABA,l-2,4-diaminobutyric acid or γ-amino-β-hydroxybutyric acid, but only slightly increased in the presence of β-alanine. These results would suggest that newly synthesized GABA and newly taken up GABA localized individually in different pools, which might localize either in different nerve terminals or separately in the same nerve terminal.     

Silica nanoparticles induce oxidative stress and inflammation of human peripheral blood mononuclear cells

In the present study, the effects of 10- or 100-nm silica oxide (SiO₂) NPs on human peripheral blood mononuclear cells (PBMC) were examined. Cytotoxic effects and oxidative stress effects, including glutathione (GSH) depletion, the formation of protein radical species, and pro-inflammatory cytokine responses, were measured. PBMC exposed to 10-nm NP concentrations from 50 to 4,000 ppm showed concentration-response increases in cell death; whereas, for 100-nm NPs, PBMC viability was not lost at <500 ppm. Interestingly, 10-nm NPs were more cytotoxic and induced more oxidative stress than 100-nm NPs. Immunoelectron micrographs show the cellular distribution of GSH and NPs. As expected based on the viability data, the 10-nm NPs disturbed cell morphology to a greater extent than did the 100-nm NPs. Antibody to the radical scavenger, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), was used for Western blot analysis of proteins with radicals; more DMPO proteins were found after exposure to 10-nm NPs than 100-nm NPs. Examination of cytokines (TNF-α, IL-1ra, IL-6, IL-8, IL-1β, and IFN-γ) indicated that different ratios of cytokines were expressed and released after exposure to 10- and 100-nm NPs. IL-1β production was enhanced by 10- and 100-nm NPs;, the cytotoxicity of the NPs was associated with an increase in the IL-1β/IL-6 ratio and 100-nm NPs at concentrations that did not induce loss of cell viability enhanced IL-1β and IL-6 to an extent similar to phytohemagglutinin (PHA), a T cell mitogen. In conclusion, our results indicate that SiO₂ NPs trigger a cytokine inflammatory response and induce oxidative stress in vitro, and NPs of the same chemistry, but of different sizes, demonstrate differences in their intracellular distribution and immunomodulatory properties, especially with regard to IL-1β and IL-6 expression.