Acid challenge exacerbates activation of matrix metalloproteinases in permanent teeth undergoing radiotherapy.

The aim of this study was to investigate the effect of acid challenge on the activation of matrix metalloproteinases (MMPs) in the Dentinoenamel junction of primary and permanent teeth submitted to radiotherapy. For this purpose, a total of 178 dental fragments obtained from molars were used, and randomly divided into 2 groups (primary and permanent teeth) / 4 experimental subgroups (irradiated and non-irradiated, demineralized and non-demineralized). The fragments were exposed to radiation, with a dose fraction of 2 Gy, for 5 consecutive days, until a total dose of 60 Gy was reached, with a total of 30 cycles, for 6 weeks. To determine the activity of MMPs on the dentinoenamel junction (DEJ), in situ zymography assays on 0.6mm dental fragments were performed. To assess whether MMP activity would be impacted by an acidic environment, the fragments were placed in a demineralizing solution (pH of 4.8). The finding was that irradiation activated MMPs in DEJ and these effects were more evident in permanent when compared with primary teeth. When the effect of an acid challenge on MMPs activity was investigated, demineralization was observed not to increase MMPs activity in non-irradiated teeth, but it did increase MMPs activity in irradiated teeth. In conclusion, an acid challenge was found to exacerbate activation of MMPs in DEJ of permanent teeth submitted to irradiation, but not in primary teeth.

Light-stimulated T. thermophilus two-domain LPMO9H: Low-resolution SAXS model and synergy with cellulases.

Lytic polysaccharide monooxygenases (LPMOs), monocopper enzymes that oxidatively cleave recalcitrant polysaccharides, have important biotechnological applications. Thermothelomyces thermophilus is a rich source of biomass-active enzymes, including many members from auxiliary activities family 9 LPMOs. Here, we report biochemical and structural characterization of recombinant TtLPMO9H which oxidizes cellulose at the C1 and C4 positions and shows enhanced activity in light-driven catalysis assays. TtLPMO9H also shows activity against xyloglucan. The addition of TtLPMO9H to endoglucanases from four different glucoside hydrolase families (GH5, GH12, GH45 and GH7) revealed that the product formation was remarkably increased when TtLPMO9H was combined with GH7 endoglucanase. Finally, we determind the first low resolution small-angle X-ray scattering model of the two-domain TtLPMO9H in solution that shows relative positions of its two functional domains and a conformation of the linker peptide, which can be relevant for the catalytic oxidation of cellulose and xyloglucan.

Photobiomodulation increases cell viability via AKT activation in an in vitro model of diabetes induced by glucose neurotoxicity.

Peripheral neuropathy (PN) is a serious complication of diabetes mellitus (DM) and is known to be resistant to conventional treatment. Photobiomodulation (PBM) is demonstrated to be effective in treating PN and in protecting nerve fiber damage. To better understand the mechanisms underlying the regenerative effects of PBM on diabetic neuropathy, we conducted a study in an in vitro model of diabetes induced by glucose neurotoxicity. Neuro 2A cells (1 × 104 cells/ well; N2A) were cultured in Minimum Essential Medium (MEM) supplemented with high glucose concentrations (100 mM) for 48 h and after the incubation period were submitted to either one or three consecutive applications of PBM, once a day (low-level InGaAlP, continuous wave mode, 660 nm, 30 mW, 1.6 J/cm2, 15 s, per well). Cell viability was measured by MTT method, neurotoxicity by LDH release, neurite outgrowth was evaluated through morphometric analysis, and AKT/ERK protein expression levels were assessed by western blotting. Results demonstrate that PBM increased N2A viability as well as induced neurogenesis observed by the increase in neurite outgrowth being this effect modulated by AKT activation. Data obtained herein reinforce the regenerative potential of PBM in the treatment of PN and strongly suggests that phototherapy should be considered adjuvant in the treatment of diabetes.

Lysosomal and mitochondrial permeabilization mediates zinc(II) cationic phthalocyanine phototoxicity.

In order to find a novel photosensitizer to be used in photodynamic therapy for cancer treatment, we have previously showed that the cationic zinc(II) phthalocyanine named Pc13, the sulfur-linked dye 2,9(10),16(17),23(24)-tetrakis[(2-trimethylammonium) ethylsulfanyl]phthalocyaninatozinc(II) tetraiodide, exerts a selective phototoxic effect on human nasopharynx KB carcinoma cells and induces an apoptotic response characterized by an increase in the activity of caspase-3. Since the activation of an apoptotic pathway by chemotherapeutic agents contributes to the elimination of malignant cells, in this study we investigated the molecular mechanisms underlying the antitumor action of Pc13. We found that after light exposure, Pc13 induced the production of reactive oxygen species (ROS), which are mediating the resultant cytotoxic action on KB cells. ROS led to an early permeabilization of lysosomal membranes as demonstrated by the reduction of lysosome fluorescence with acridine orange and the release of lysosomal proteases to cytosol. Treatment with antioxidants inhibited ROS generation, preserved the integrity of lysosomal membrane and increased cell proliferation in a concentration-dependent manner. Lysosome disruption was followed by mitochondrial depolarization, cytosolic release of cytochrome C and caspases activation. Although no change in the total amount of Bax was observed, the translocation of Bax from cytosol to mitochondria, the cleavage of the pro-apoptotic protein Bid, together with the decrease of the anti-apoptotic proteins Bcl-XL and Bcl-2 indicated the involvement of Bcl-2 family proteins in the induction of the mitochondrial pathway. It was also demonstrated that cathepsin D, but not caspase-8, contributed to Bid cleavage. In conclusion, Pc13-induced cell photodamage is triggered by ROS generation and activation of the mitochondrial apoptotic pathway through the release of lysosomal proteases. In addition, our results also indicated that Pc13 induced a caspase-dependent apoptotic response, being activation of caspase-8, -9 and -3 the result of a post-mitochondrial event.

Caspase-3 activation and increased procollagen type I in irradiated hearts.

The caspase-3-cleaved presence was evaluated in this study in the heart of irradiated rats, during the decline of ventricular function. Female Wistar rats were irradiated with a single dose of radiation (15 Gy) delivered directly to the heart and the molecular, histological and physiological evaluations were performed at thirteen months post-irradiation. The expressions of procollagen type I, TGF-ß1 and caspase-3-cleaved were analyzed using Western blotting. Cardiac structural and functional alterations were investigated by echocardiography and electron microscopy. In the irradiated group, the levels of procollagen type I, TGF-ß1 and caspase-3-cleaved are increased. Significant histological changes (degeneration of heart tissue and collagen deposition) and functional (reduced ejection fraction) were observed. Data suggest that the cardiac function decline after exposure to ionizing radiation is related, in part, to increased collagen and increased caspase-3-cleaved.

Ultrasound-assisted lipase-catalyzed transesterification of soybean oil in organic solvent system.

This work reports the transesterification of soybean oil with ethanol using two commercial immobilized lipases under the influence of ultrasound irradiation. The experiments were performed in an ultrasonic water bath, following a sequence of experimental designs to assess the effects of temperature, enzyme and water concentrations, oil to ethanol molar ratio and output irradiation power on the reaction yield. Results show that ultrasound-assisted lipase-catalyzed transesterification of soybean oil with ethanol might be a potential alternative route to conventional alkali-catalyzed method, as high reaction yields (~90 wt.%) were obtained at mild irradiation power supply (~100 W), and temperature (60 °C) in a relatively short reaction time, 4h, using Lipozyme RM IM as catalyst. The repeated use of the catalyst under the optimum experimental condition resulted in a decay in both enzyme activity and product conversion after two cycles. The use of Novozym 435 led to lower conversions (about 57%) but the enzyme activity was stable after eight cycles of use, showing, however, a reduction in product conversion after the forth cycle.

Kinetics of ultrasound-assisted lipase-catalyzed glycerolysis of olive oil in solvent-free system.

This work reports experimental kinetic data of solvent-free glycerolysis of olive oil using a commercial immobilized lipase (Novozym 435) under the influence of ultrasound irradiation. The experiments were performed in a mechanically stirred reactor under ultrasound irradiation, evaluating the effects of temperature (50-70 °C), enzyme concentration (2.5-10 wt%) and glycerol to oil molar ratio (0.8:1-3:1). Results show that ultrasound-assisted lipase-catalyzed glycerolysis might be a potential alternative route to conventional methods, as high contents of reaction products, especially monoglycerides, were achieved at mild irradiation power supply (~130 W) and temperature, in a relatively short reaction time (2h) and low enzyme content (7.5 wt%). To completeness, two simplified kinetic modeling approaches, based on the ordered-sequential bi bi mechanism and reaction stoichiometry, were employed to represent the experimental data, thus allowing a better understanding of the reaction kinetics.

Deleterious MnSOD signals lead to abnormal breast cell proliferation by radiation and estrogen exposure.

Manganese superoxide dismutase (MnSOD) seems to have a pivotal role in maintaining the normal phenotype by suppressing cell growth through blocking the entrance of quiescent cells into the cell cycle. MnSOD protein expression has been shown to be dysregulated in malignant cells. A well-established experimental breast epithelial cell cancer model was used to observe the relationship in the presence or absence of such protein and the phenotype of the cells. This model was derived from the spontaneously immortalized breast epithelial cell line MCF-10F, which was transformed with estrogen and radiation. The results of this study showed that deleterious expression of MnSOD enhanced the malignant phenotype demonstrated by cell cycle protein expression changes. Thus, the malignant cell line, called Alpha5, which had high levels of MnSOD protein expression, maintained a similar phenotype to the normal cell line MCF-10F. The cell cycle arrest observed in G1 phase of the Alpha5 cell line was induced by p16 protein expression which has been shown to inhibit the Cyclin D1/CdK4 complex explaining such arrest. It can be concluded from these studies that SOD expression, played a critical role in free radical detoxification and it is directly correlated with the cell cycle, defining one of the most important characteristics of tumor cells, namely cell growth and proliferation. These findings are in agreement with the hypothesis that MnSOD plays a role as a possible tumor suppressor gene. Furthermore, this work is a contribution to understanding the possible changes that occur in α-particle irradiated cells, sensitized with estrogen, due to the presence of superoxide dismutase scavenger that could have significant implications in the design of clinical radiotherapeutic protocols.

Lipase-catalyzed diacylglycerol production under sonochemical irradiation.

The present paper describes a protocol for production of diacylglycerol by the partial hydrolysis of soybean oil catalyzed by lipase under ultrasound irradiation. Better yields and shorter reaction times were obtained under sonication as compared to the thermal process.

Effect of GaAlAs laser irradiation on enzyme activity.

OBJECTIVE: The aim of this study was to determine the influence of laser irradiation on enzyme activity. BACKGROUND DATA: Enzymes are catalysts of extraordinary efficiency, able to accelerate reactions by manifold. Enzyme laser light activation is currently a fast-growing field and a large number of studies have been produced. MATERIALS AND METHODS: Liquid CNPG amylase and control serum (Qualitrol 1H) were used in the experiments. Laboratory analysis of alpha-amylase was performed on two sample groups: (i) E + S and (ii) E + S + L, in six repetitions per irradiation dose. Group 2 was irradiated with gallium-aluminum-arsenide (GaAlAs) 904 nm at doses of 0.01, 0.1, 0.5, and 1 J/cm(2). Enzyme activity was read using a spectrophotometer equipped with a thermostatic chamber capable of precise absorbance measurement at 405 nm. RESULTS: The results were analyzed with the Student's t-test, and the percentage of enzyme activity was determined. Photomodulation of alpha-amylase activity by GaAlAs laser was analyzed following irradiation with different doses. Irradiation doses from 0.01 to 1 J/cm(2) led to differences in enzyme activity: 0.01 J/cm(2) (0.10%), 0.1 J/cm(2) (13.44%), 0.5 J/cm(2) (12.57%), and 1 J/cm(2) (-6.10%). CONCLUSION: Irradiation doses of 0.1 J/cm(2) and 0.5 J/cm(2) led to statistically significant increases in enzyme activity in comparison to the control. The similar curves of the effects of temperature and pH on enzymatic activity observed in this study suggest that laser irradiation also possess an optimum dose to modulate the enzymatic activity. That is, enzymes have an optimum laser dose (or range) at which their activity is maximal, whereas at higher or lower doses activity decreases.

Biostimulation of Na,K-ATPase by low-energy laser irradiation (685 nm, 35 mW): comparative effects in membrane, solubilized and DPPC:DPPE-liposome reconstituted enzyme.

OBJECTIVE: The aim of the present work was to investigate the effect of low-energy laser irradiation (685 nm, 35 mW) on the ATPase activity of the different forms of the Na,K-ATPase. METHODS: Membrane-bound and solubilized (alphabeta)(2) form of Na,K-ATPase was obtained from the dark red outer medulla of the kidney and proteoliposomes of DPPC:DPPE and Na,K-ATPase was prepared by the co-solubilization method. Irradiations were carried out at 685 nm using an InGaAIP diode laser. RESULTS: The ATPase activity of the membrane fraction was not altered with exposition to irradiation doses between 4 and 24 J/cm(2). However, with irradiation doses ranging from 32 to 40 J/cm(2), a 28% increase on the ATPase activity was observed while when using up to 50 J/cm(2) no additional enhancement was observed. When biostimulation was done using the solubilized and purified enzyme or the DPPC:DPPE-liposome reconstituted enzyme, an increase of about 36-40% on the ATPase activity was observed using only 4-8 J/cm(2). With irradiation above these values (24 J/cm(2)) no additional increase in the activity was observed. These studies revealed that the biostimulation of ATPase activity from different forms of the Na,K-ATPase is dose dependent in different ranges of irradiation exposure. The stimulation promoted by visible laser doses was modulated and the process was reverted after 2 h for the enzyme present in the membrane and after about 5 h for the solubilized or the reconstituted in DPPC:DPPE-liposomes.

alpha- and epsilon-protein kinase C activity during smooth muscle cell apoptosis in response to gamma-radiation.

The use of gamma-radiation in treatment of pelvic cancer is associated with injury of healthy surrounding tissues and disorders of intestinal motility; however, the cellular mechanisms involved are unclear. We tested the hypothesis that exposure of visceral smooth muscle cells (SMCs) to gamma-radiation induces apoptosis via activation of specific protein kinase C (PKC) isoforms. Cultured SMCs and slices from guinea pig ileum smooth muscle longitudinal layer (GPISMLL) were exposed to 10 to 50 Gy. Flow cytometry in gamma-radiated SMCs showed increased percentage of cells in the sub-G(0)/G(1) phase, a hallmark of apoptosis. gamma-Radiation-induced reduction in cell survival was partially but significantly alleviated with the PKC inhibitors. Sections of gamma-irradiated GPISMLL showed DNA fragmentation and apoptotic bodies analyzed by the terminal deoxynucleotidyl transferase dUTP nick-end labeling method, whereas the plasma and nuclear membranes were preserved. Confocal microscopy in gamma-radiated SMCs labeled with annexin V-fluorescein showed an increase in apoptotic cells and phosphatidylserine externalization. Contraction of GPISMLL strips in response to KCl and acetylcholine was reduced in tissues exposed to 30 and 50 Gy. gamma-Radiation of GPISMLL caused an increase in PKC activity in the particulate fraction, a decrease in the cytosolic fraction, and increased particulate/cytosolic PKC activity ratio. Western blot analysis revealed significant amounts of alpha- and epsilon-PKC in the cytosolic fraction of control GPISMLL. gamma-Radiation caused an increase in the amount of alpha- and epsilon-PKC in the particulate fraction and a decrease in the cytosolic fraction. Data suggest that gamma-radiation induces apoptosis, growth arrest, and contractile dysfunction in visceral SMCs of GPISMLL via activation and translocation of alpha- and epsilon-PKC isoforms.

Photodynamic activity of a new sensitizer derived from porphyrin-C60 dyad and its biological consequences in a human carcinoma cell line.

The photokilling activity of a porphyrin-C(60) (P-C(60)) dyad was evaluated on a Hep-2 human larynx-carcinoma cell line. This study represents the first evaluation of a dyad, with high capacity to form a photoinduced charge-separated state, to act as agent to inactivate cells by photodynamic therapy (PDT). Cell treatment was carried out with 1 microM P-C(60) incorporated into liposomal vesicles. No dark cytotoxicity was observed using 1 microM P-C(60) concentration and during long incubation time (24h). The uptake of sensitizer into Hep-2 was studied at different times of incubation. Under these conditions, a value of 1.5 nmol/10(6)cells was found after 4h of incubation showing practically no change even after 24h. The cell survival after irradiation of the cells with visible light was dependent upon light exposure level. A high photocytotoxic effect was observed for P-C(60), which inactivated 80% of the cells after 54 J/cm(2) of irradiation. Moreover, the dyad kept a high photoactivity even under argon atmosphere. Thus, depending on the microenvironment where the sensitizer is localized, this compound could produce a biological photodamage through either a (1)O(2)-mediated photoreaction process or a free radical mechanism under low oxygen concentration. The mechanism of cell death was analyzed by Hoechst-33258, toluidine blue staining, TUNEL and DNA fragmentation. Cell cultures treated for 24h with P-C(60) and irradiated with a dose of 54 J/cm(2) showed a great amount of apoptotic cells (58%). Moreover, changes in cell morphology were analyzed using fluorescence microscopy with Hoechst-33258 under low oxygen concentration. Under this anaerobic condition, necrotic cellular death predominated on apoptotic pathway. There were more apoptotic cells under air irradiation condition than under argon irradiation condition. To determine the apoptotic pathway, caspase-3 activation was studied by caspase-3 activity detection kits. The last results showed that P-C(60) induced apoptosis by caspase-3-dependent pathway. These results indicated that molecular dyad, which can form a photoinduced charge-separated state, is a promising model for phototherapeutic agents and they have potential application in cell inactivation by PDT.

Signalling responses linked to betulinic acid-induced apoptosis are antagonized by MEK inhibitor U0126 in adherent or 3D spheroid melanoma irrespective of p53 status.

MEK1/2 inhibitors like U0126 can potentiate or antagonize the antitumor activity of cytotoxic agents such as cisplatin, paclitaxel or vinblastine, depending on the drug or the target cells. We now investigated whether U0126, differentially regulates melanoma signaling in response to UV radiation or betulinic acid, a drug lethal against melanoma. This report shows that U0126 inhibits early response (ERK) kinase activation and cyclin A expression in wt p53 C8161 melanoma exposed to either UV radiation or betulinic acid. However, U0126 does not protect from UV damage, but counteracts betulinic acid-mediated apoptosis in the same cells. Protection from the latter drug by joint treatment with U0126 was also evident in wt p53 MelJuso melanoma and mutant p53 WM164 melanoma. The latter cells were the most responsive to betulinic acid, showing a selective decline in the cdk4 protein, without a comparable change in other key cell cycle proteins like cdc2, cdk2, cdk7 or cyclin A, prior to apoptosis-associated PARP fragmentation. Laser scanning cytometry also showed that betulinic acid induced a significant increase in chromatin condensation in WM164 melanoma irrespective of whether they were in adherent form or as multicellular spheroids. All these betulinic acid-induced changes were counteracted by U0126. Our data show for the first time that (a) cdk4 protein is an early target of betulinic acid-induced apoptosis and (b) unrestricted ERK signaling favours betulinic acid-induced apoptosis, but this is counteracted by U0126, partly through counteracting chromatin condensation and restoring Akt activation decreased by betulinic acid treatment.

Light regulation of sucrose-phosphate synthase activity in the freezing-tolerant grass Deschampsia antarctica.

Deschampsia antarctica, a freezing-tolerant grass that has colonized the Maritime Antarctic, has an unusually high content of sucrose (Suc) in leaves, reaching up to 36% of dry weight. Suc accumulation has often been linked with increased activity of sucrose phosphate synthase (SPS; EC: 2.4.1.1.14). SPS, a key enzyme in sucrose biosynthesis, is controlled by an intricate hierarchy of regulatory mechanisms including allosteric modulators, reversible covalent modification in response to illumination, and transcriptional regulation. We hypothesized that during long day conditions in the Antarctic summer D. antarctica can maintain high SPS activity longer by indirect light regulation, thereby leading to a high sucrose accumulation in the leaves. The objectives of this study were to investigate a possible indirect light regulation of SPS activity and the effect of cold and day length on transcriptional and protein level of SPS in D. antarctica. Although SPS activity did not display an endogenous rhythm of activity in continuous light, activation of SPS at the end of the dark period was observed in D. antarctica. This activation of SPS is possibly controlled by covalent modification, because it was inhibited by okadaic acid while the SPS protein level did not significantly change. The highest SPS activity increase was observed after 21 days of cold-acclimation under long day conditions. This increased activity was not related to an increase in SPS gene expression or protein content. High SPS activity in cold long days leading to hyper accumulation of Suc appears to be among the features that permit D. antarctica to survive in the harsh Antarctic conditions.

Ultraviolet exposure of thymocytes: selective inhibition of apoptosis.

PURPOSE: To evaluate selective effects of ultraviolet (UV) irradiation on spontaneous and induced apoptosis in freshly extracted mice thymocytes. MATERIALS AND METHODS: Cells were exposed to UV radiation with emission peaks of 365 nm (UVA) exposures of 1620-10200 J m(-2), of 312 nm (UVB) exposures of 34-1620 J m(-2) or of 254 nm (UVC) exposures of 1.5-1620 J m(-2), and incubated for 5.5 h with or without hydrocortisone, phorbol-12-myristate-13-acetate or anti-Fas antibody. Additionally, cells were irradiated with gamma-rays (5 Gy) before UVB exposure (408 J m(-2)) at different times. Apoptosis was quantified by DNA fragmentation. RESULTS: Up to an irradiation of 5000 J m(-2), UVA exposure did not show any effect on thymocyte apoptosis, while at 10200 J m(-2) irradiation, considerable DNA fragmentation was observed. In contrast, UVB and UVC irradiation clearly inhibited natural and cortisone-induced apoptosis. Moreover, UVB inhibited apoptosis triggered by phorbol-12-myristate-13-acetate and gamma-irradiation, but not by anti-Fas antibody. CONCLUSIONS: The response of mouse thymocytes in culture to UV irradiation strongly depends on the wavelength used. It is suggested that either a survival or an apoptotic pathway occurs depending on the physiological state of the cell, spectral composition of the UV light and cell type. The possible involvement of extracellular signal-regulated kinase and stress-activated protein kinase/c-Jun N-terminal kinase in the apoptotic pathway is discussed.

Increased activity and involvement of caspase-3 in radiation-induced apoptosis in neural cells precursors from developing rat brain.

Using primary cultures of neural precursor cells of cortex from developing rat brain, we demonstrated the involvement of caspase-3 in the apoptotic process induced by gamma irradiation. The precursor nature of cells was confirmed by nestin and GFAP immunoreactivity and by the capacity of differentiation in neuronal and glial cells after 5 days in culture. Neural precursors were irradiated with single doses ranging from 0.1 to 4Gy. Cellular death, determined 24 h post-irradiation (pi) was dose-dependent and the induction of apoptosis was confirmed by nuclear condensation, DNA fragmentation and hypodiploid DNA peak represented by the "sub G1" region. For the higher doses, apoptosis was evident after 4-6 h pi and increased during 24 h. Caspase-3 activity increased with doses and was maximal at 4-6 h pi with 3Gy and remained similar with 4Gy. The protection from radiation-induced apoptosis by caspase-3 inhibitor, zDEVD-fmk, confirmed that this enzyme is involved in the apoptotic mechanism in this system. The possibility of using this tissue culture system for studying the effects of ionizing radiation on morphological and molecular differentiation was considered.

N-Acetylcysteine enhances UV-mediated caspase-3 activation, fragmentation of E2F-4, and apoptosis in human C8161 melanoma: inhibition by ectopic Bcl-2 expression.

Redox imbalance due to oxidative stress or excessive antioxidant levels can alter apoptotic responses. Recently, antioxidants like N-acetylcysteine (NAC) were reported to inhibit H(2)O(2)-mediated necrotic cell death, although they were inactive against apoptosis induced by other agents like etoposide. NAC was also found to kill preferentially tumor cells compared to normal fibroblasts at 20-50mM, but these concentrations are lethal to normal splenocytes. We now demonstrate that 10mM NAC, a non-toxic concentration, can enhance the UV radiation-mediated apoptosis of human C8161 melanoma cells. Compared to treatment with UV radiation alone, combination treatment with NAC doubled the ratio of activated caspase-3 to pro-caspase-3 and produced greater fragmentation of the retinoblastoma protein and the E2F-4 transcription factor without affecting the E2F-1 protein. These effects of joint NAC-UV radiation treatment were counteracted by the overexpression of the bcl-2 gene. To our knowledge, this report is the first to: (i) demonstrate a synergy between DNA-damaging agents, like UV radiation, and antioxidants, like NAC, and (ii) show that a Bcl-2-inhibitable E2F-4 fragmentation occurs concurrently with caspase-3 activation and apoptosis.

Influence of temperature upon effects of crotoxin and gamma-irradiated crotoxin at rat neuromuscular transmission.

The influence of temperature upon the effects of crotoxin (CTX), from Crotalus durissus terrificus venom, and gamma-irradiated (60Co, 2000 Gy) crotoxin (iCTX) was studied in rat neuromuscular transmission 'in vitro'. Indirect twitches were evoked in the phrenic-diaphragm preparation by supramaximal strength pulses with a duration of 0.5 ms and frequency of 0.5 Hz. The phospholipase A(2) (PLA(2)) enzymatic activity of CTX and iCTX was assayed against phosphadityl choline in Triton X-100. At 27 degrees C, CTX (14 microg/ml) did not affect the amplitude of indirectly evoked twitches. However, at 37 degrees C, CTX induced a time-dependent blockade of the neuromuscular transmission that started at 90 min and was completed within 240 min. iCTX (14 microg/ml) was inneffective on the neuromuscular transmission either at 27 or 37 degrees C. The PLA(2) enzymatic activity of CTX at 37 degrees C was 84 and that at 27 degrees C was 27 micromol fatty acid released/min/mg protein, and that of the iCTX at 37 degrees C was 39 micromol fatty acid released/min/mg protein. Thus, it was concluded that the mechanism of detoxification of CTX by gamma radiation at the neuromuscular level relies on the loss of its PLA(2) enzymatic activity.

Phosphorylation of rod outer segment proteins modulates phosphatidylethanolamine N-methyltransferase and phospholipase A2 activities in photoreceptor membranes.

The activities of enzymes involved in lipid metabolism--phospholipase A2 (PLA2) and phosphatidylethanolamine N-methyltransferase (PE N-MTase)--were found to be differently affected by pre-incubation of rod outer segments (ROS) under protein phosphorylating or dephosphorylating conditions. Exposure to cAMP-dependent protein kinase (PKA), under dark or light conditions, produced a significant increase in PE N-MTase activity, whereas PLA2 activity decreased. Under standard protein kinase C (PKC) phosphorylating conditions in light, PE N-MTase activity was stimulated and PLA2 activity was not affected. When the assays were performed in the dark, both enzymatic activities were unaffected when compared to the corresponding controls. Incubation of ROS membranes in light in the presence of PKC activators phorbol 12,13-dibutyrate (PDBu) and dioctanoylglycerol (DOG) resulted in the same pattern of changes in enzyme activities as described for standard PKC phosphorylating condition. Pre-incubation of membranes with the PKC inhibitor H-7 reduced the stimulation of PDBu on PE N-MTase activity, and had no effect on PLA2 activity in ROS membranes incubated with the phorbol ester. Pre-treatment of isolated ROS with alkaline phosphatase resulted in decreased PE N-MTase activity and produced a significant stimulation of PLA2 activity under dark as well as under light conditions when compared to the corresponding controls. These findings suggest that ROS protein phosphorylation and dephosphorylation modulates PE N-MTase and PLA2 activities in isolated ROS, and that these activities are independently and specifically modulated by particular kinases. Furthermore, dephosphorylation of ROS proteins has the opposite effect to that produced by protein phosphorylation on the enzymes studied.