Anti-Mayaro virus activity of a hydroethanolic extract from Fridericia chica (Bonpl.) L. G. Lohmann leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: Mayaro fever is a neglected tropical disease. The region of the most significant circulation of the Mayaro virus (MAYV) is the Amazon rainforest, situated in remote areas that are difficult to access and where medicine is scarce. Thus, the regional population uses plants as an alternative for the treatment of various diseases. Fridericia chica is an endemic plant of tropical regions used in traditional medicine to treat fever, malaise, inflammation, and infectious diseases such as hepatitis B. However, its antiviral activity is poorly understood. AIM OF THE STUDY: This study aimed to investigate the anti-MAYV activity of the hydroethanolic extract of the leaves of Fridericia chica (HEFc) in mammalian cells and its possible mechanism of action. MATERIALS AND METHODS: The antiviral activity of HEFc was studied using Vero cell lines against MAYV. The cytotoxicity and antiviral activity of the extract were evaluated by the 3-(4, 5- dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. The overall antiviral activity was confirmed by the plaque forming units (PFU) method. Then, the effects of HEFc on MAYV multiplication kinetics, virus adsorption, penetration, and post-penetration, and its virucidal activity were determined in Vero cells using standard experimental procedures. RESULTS: HEFc exerted a effect against viral infection in Vero cells at a non-cytotoxic concentration, and no virion was detected in the supernatant in a dose-dependent and selective manner. HEFc inhibited MAYV in the early and late stages of the viral multiplication cycle. The extract showed significant virucidal activity at low concentrations and did not affect adsorption or viral internalization stages. In addition, HEFc reduced virions at all post-infection times investigated. CONCLUSIONS: HEFc has good antiviral activity against MAYV, acting directly on the viral particles. This plant extract possesses an excellent and promising potential for developing effective herbal antiviral drugs.

Synthesis of silver nanoparticles using Alpinia officinarum rhizome extract induces apoptosis through down-regulating Bcl-2 in human cancer cells.

In this study, silver nanoparticles were synthesized using Alpinia officinarum rhizome extract via an eco-friendly green synthesis method. The silver nanoparticles (AO-AgNPs) were characterized by UV-Vis spectrometry, scanning electron microscopy, energy-dispersive X-ray spectroscopy, and dynamic light scattering. Further, the cytotoxic and apoptotic effects of AO-AgNPs were investigated in human cancer cells with different tissue origins via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and flow cytometric analyses, respectively. The expression levels of anti-apoptotic Bcl-2 protein were evaluated via a real-time polymerase chain reaction. The synthesized AO-AgNPs induced a significant cytotoxic effect in all tested cancer cells but not in normal cells. AO-AgNPs induced the percentage of apoptotic cells and reduced the levels of anti-apoptotic Bcl-2 mRNA levels in cancer cells. These results demonstrate the potential application of AO-AgNPs in cancer treatment.

In vitro Evaluation of Selective Cytotoxic Activity of Chaerophyllum macropodum Boiss. on Cultured Human SH-SY5Y Neuroblastoma Cells.

Neuroblastoma is the most common solid tumor in children. New treatment approaches are needed because of the harmful side effects and costs of the methods used in the treatment of neuroblastoma. Medicinal and aromatic plants are important for new treatment approaches due to their minimal side effects and economic advantages. Therefore, the present study was carried out to examine the cytotoxic effect of Chaerophyllum macropodum extract on human neuroblastoma (SH-SY5Y) and fibroblast (HDFa) cell lines. 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase release (LDH) assays were used to determine the cytotoxic effect of C. macropodum. The extracts were analyzed for their phenolic content by HPLC-PDA. Major components were determined as 63.600% o-coumaric acid, 15.606% catechine hydrate, 8.713% rosmarinic acid, 4.376% clorogenic acid, and 3.972% salicylic acid. The obtained results from cytotoxicity testing revealed that C. macropodum exerted a significant cytotoxic effect on human neuroblastoma cells at all tested concentrations (p < 0.05). But it did not lead to any cytotoxic potential on human fibroblasts. As a result, the obtained data clearly revealed C. macropodum exerted a selective cytotoxic action on neuroblastoma cells for the first time.

Bromide impairs the circadian clock and glycolytic homeostasis via disruption of autophagy in rat H9C2 cardiomyocytes.

BACKGROUND: Trace elements function as essential cofactors that are involved in various biochemical processes in mammals. Autophagy is vital for nutrient supplement, which is an important Zeitegber for the circadian homeostasis in heart. Here, we considered the possibility that autophagy, as well as the cardiomyocyte clock and glycolysis are interlinked. Detrimental effects were observed when cardiac system is exposed to bromine containing drugs. This study investigated the effects and mechanisms of bromide on the circadian clock and glycolytic metabolism of H9C2 cardiomyocytes. RESULTS: In the present study, bromide does not affect cell viability and apoptosis of H9C2 cardiomyocytes. Bromide dampens the clock and glycolytic (Hk2 and Pkm2) gene expression rhythmicity in a dose-dependent manner. Additionally, bromide inhibits autophagic process in H9C2 cardiomyocytes. In contrast, rapamycin (an autophagy inducer) dramatically restores the inhibitory effect of NaBr on the mRNA expression levels of clock genes (Bmal1, Cry1 and Rorα) and glycolytic genes (Hk2 and Pkm2). CONCLUSIONS: Our results reveal that bromide represses the clock and glycolytic gene expression patterns, partially through inhibition of autophagy.

Inorganic Salts and Antimicrobial Photodynamic Therapy: Mechanistic Conundrums?

We have recently discovered that the photodynamic action of many different photosensitizers (PSs) can be dramatically potentiated by addition of a solution containing a range of different inorganic salts. Most of these studies have centered around antimicrobial photodynamic inactivation that kills Gram-negative and Gram-positive bacteria in suspension. Addition of non-toxic water-soluble salts during illumination can kill up to six additional logs of bacterial cells (one million-fold improvement). The PSs investigated range from those that undergo mainly Type I photochemical mechanisms (electron transfer to produce superoxide, hydrogen peroxide, and hydroxyl radicals), such as phenothiazinium dyes, fullerenes, and titanium dioxide, to those that are mainly Type II (energy transfer to produce singlet oxygen), such as porphyrins, and Rose Bengal. At one extreme of the salts is sodium azide, that quenches singlet oxygen but can produce azide radicals (presumed to be highly reactive) via electron transfer from photoexcited phenothiazinium dyes. Potassium iodide is oxidized to molecular iodine by both Type I and Type II PSs, but may also form reactive iodine species. Potassium bromide is oxidized to hypobromite, but only by titanium dioxide photocatalysis (Type I). Potassium thiocyanate appears to require a mixture of Type I and Type II photochemistry to first produce sulfite, that can then form the sulfur trioxide radical anion. Potassium selenocyanate can react with either Type I or Type II (or indeed with other oxidizing agents) to produce the semi-stable selenocyanogen (SCN)2. Finally, sodium nitrite may react with either Type I or Type II PSs to produce peroxynitrate (again, semi-stable) that can kill bacteria and nitrate tyrosine. Many of these salts (except azide) are non-toxic, and may be clinically applicable.

Molecular cloning, characterization, expression and enzyme activity of catalase from planarian Dugesia japonica in response to environmental pollutants.

Catalase (CAT) is an important antioxidant enzyme that protects aerobic organisms against oxidative damage by degrading hydrogen peroxide to oxygen and water. CAT mRNAs have been cloned from many species and employed as useful biomarkers of oxidative stress. In the present study, we cloned the cDNA sequence of CAT gene from freshwater planarian Dugesia japonica (designated as DjCAT) by means of RACE method. Sequence analysis and multiple alignment jointly showed that the full-length cDNA sequence consists of 1734 nucleotides, encoding 506 amino acids. Three catalytic amino acid residues of His71, Asn144 and Tyr354, two CAT family signature sequences of a proximal active site signature (60FDRERIPERVVHAKGGGA77) and a heme-ligand signature motif (350RLFSYRDTQ358) are highly conserved, suggesting that the DjCAT belongs to the NADPH and heme-binding CAT family and has similar functions. In addition, the transcriptional level of CAT gene and activity of CAT enzyme upon acute exposure of environmental pollutants glyphosate and 1-decyl-3-methylimidazolium bromide ([C10mim]Br) were investigated systematically. The variation of CAT mRNA expression in D. japonica was quantified by real-time PCR and the results indicated that it was up-regulated after exposure to glyphosate or [C10mim]Br with a dose-dependent manner but not linearly. Even though the variation trend of CAT activity upon glyphosate stress was not monotonously increased and inconsistent with that after [C10mim]Br exposure on day 1 and 3 sampling time, with the duration prolonged to day 5 they both presented a dose-dependent increase and the differences achieved extreme significance in all treated groups compared to the control. These findings suggested that DjCAT plays an important role in antioxidant defense in D. japonica, and the mRNA expression of CAT would also be used as an effective biomarker to monitor the pollution in aquatic environment just like its corresponding enzyme.

Bromide supplementation exacerbated the renal dysfunction, injury and fibrosis in a mouse model of Alport syndrome.

A seminal study recently demonstrated that bromide (Br-) has a critical function in the assembly of type IV collagen in basement membrane (BM), and suggested that Br- supplementation has therapeutic potential for BM diseases. Because salts of bromide (KBr and NaBr) have been used as antiepileptic drugs for several decades, repositioning of Br- for BM diseases is probable. However, the effects of Br- on glomerular basement membrane (GBM) disease such as Alport syndrome (AS) and its impact on the kidney are still unknown. In this study, we administered daily for 16 weeks 75 mg/kg or 250 mg/kg (within clinical dosage) NaBr or NaCl (control) via drinking water to 6-week-old AS mice (mouse model of X-linked AS). Treatment with 75 mg/kg NaBr had no effect on AS progression. Surprisingly, compared with 250 mg/kg NaCl, 250 mg/kg NaBr exacerbated the progressive proteinuria and increased the serum creatinine and blood urea nitrogen in AS mice. Histological analysis revealed that glomerular injury, renal inflammation and fibrosis were exacerbated in mice treated with 250 mg/kg NaBr compared with NaCl. The expressions of renal injury markers (Lcn2, Lysozyme), matrix metalloproteinase (Mmp-12), pro-inflammatory cytokines (Il-6, Il-8, Tnf-α, Il-1ß) and pro-fibrotic genes (Tgf-ß, Col1a1, α-Sma) were also exacerbated by 250 mg/kg NaBr treatment. Notably, the exacerbating effects of Br- were not observed in wild-type mice. These findings suggest that Br- supplementation needs to be carefully evaluated for real positive health benefits and for the absence of adverse side effects especially in GBM diseases such as AS.

Drug screening in Scn1a zebrafish mutant identifies clemizole as a potential Dravet syndrome treatment.

Dravet syndrome is a catastrophic pediatric epilepsy with severe intellectual disability, impaired social development and persistent drug-resistant seizures. One of its primary monogenic causes are mutations in Nav1.1 (SCN1A), a voltage-gated sodium channel. Here we characterize zebrafish Nav1.1 (scn1Lab) mutants originally identified in a chemical mutagenesis screen. Mutants exhibit spontaneous abnormal electrographic activity, hyperactivity and convulsive behaviours. Although scn1Lab expression is reduced, microarray analysis is remarkable for the small fraction of differentially expressed genes (~3%) and lack of compensatory expression changes in other scn subunits. Ketogenic diet, diazepam, valproate, potassium bromide and stiripentol attenuate mutant seizure activity; seven other antiepileptic drugs have no effect. A phenotype-based screen of 320 compounds identifies a US Food and Drug Administration-approved compound (clemizole) that inhibits convulsive behaviours and electrographic seizures. This approach represents a new direction in modelling pediatric epilepsy and could be used to identify novel therapeutics for any monogenic epilepsy disorder.

TRPM7 is regulated by halides through its kinase domain.

Transient receptor potential melastatin 7 (TRPM7) is a divalent-selective cation channel fused to an atypical α-kinase. TRPM7 is a key regulator of cell growth and proliferation, processes accompanied by mandatory cell volume changes. Osmolarity-induced cell volume alterations regulate TRPM7 through molecular crowding of solutes that affect channel activity, including magnesium (Mg(2+)), Mg-nucleotides and a further unidentified factor. Here, we assess whether chloride and related halides can act as negative feedback regulators of TRPM7. We find that chloride and bromide inhibit heterologously expressed TRPM7 in synergy with intracellular Mg(2+) ([Mg(2+)]i) and this is facilitated through the ATP-binding site of the channel's kinase domain. The synergistic block of TRPM7 by chloride and Mg(2+) is not reversed during divalent-free or acidic conditions, indicating a change in protein conformation that leads to channel inactivation. Iodide has the strongest inhibitory effect on TRPM7 at physiological [Mg(2+)]i. Iodide also inhibits endogenous TRPM7-like currents as assessed in MCF-7 breast cancer cells, where upregulation of SLC5A5 sodium-iodide symporter enhances iodide uptake and inhibits cell proliferation. These results indicate that chloride could be an important factor in modulating TRPM7 during osmotic stress and implicate TRPM7 as a possible molecular mechanism contributing to the anti-proliferative characteristics of intracellular iodide accumulation in cancer cells.

Effects of sulfurous, salty, bromic, iodic thermal water nasal irrigations in nonallergic chronic rhinosinusitis: a prospective, randomized, double-blind, clinical, and cytological study.

OBJECTIVES: Nasal irrigations are mentioned among the adjunctive measures for treating several sinonasal conditions. Hyperchromatic supranuclear stria (HSS) in the ciliated cells (CCs) has recently been suggested as a potential cytological marker of the anatomofunctional integrity of nasal mucosa. The aim of this study was to compare the effects of nasal irrigations with sulfurous, salty, bromic, iodic (SSBI) thermal water or isotonic sodium chloride solution (ISCS) in patients with nonallergic chronic rhinosinusitis, considering the endoscopic, functional, microbiological, and cytological evidence (including the ratio of HSS-positive CCs to total CCs [the HSS+ rate]). METHODS: In a prospective, randomized, double-blind setting, 80 patients were recruited for nasal irrigations with SSBI water or ISCS for 1 month. RESULTS: An endoscopically assessed significant clinical improvement was seen after both SSBI thermal water and ISCS irrigations. Before treatment, Staphylococcus aureus was isolated in 5 patients in the SSBI thermal water group and 4 in the ISCS group. After the nasal irrigations, there was no sign of the bacteria in either group. Only the SSBI water irrigations significantly reduced total nasal resistance, as determined by rhinomanometry. Mild nasal irritation (6 cases) and episodes of extremely limited epistaxis (5 cases) were only reported after SSBI thermal water irrigations. Neither type of nasal irrigation significantly increased the mean HSS+ rate at cytological control after 1 month. CONCLUSIONS: Both types of nasal irrigation improved the endoscopic and microbiological features of patients with nonallergic chronic rhinosinusitis, whereas only SSBI irrigations significantly reduced total nasal resistance. Further investigations are needed based on longer treatments and follow-up periods to establish whether the HSS+ rate is useful for monitoring clinical improvements in chronic rhinosinusitis treated with nasal irrigations.

[The clinical efficiency and safety of iodide-bromine balneotherapy in patients with coronary heart disease concurrent with chronic obstructive pulmonary disease].

AIM: to evaluate the efficiency of iodide-bromine balneotherapy (IBB) in patients with coronary heart disease (CHD) concurrent with chronic obstructive pulmonary disease (COPD). SUBJECTS AND METHODS: Sixty-six patients with CHD, Functional Class HII stable angina on exertion concurrent with mild and moderate COPD were examined. Group 1 included 36 patients with CHD concurrent with mild and moderate COPD (a study group); Group 2 consisted of 30 patients (a control group). The groups were matched for age, gender, and concomitant abnormality. The patients from both groups undergone a complex clinical and instrumental study (clinical and biochemical study, echocardiography, 24-hour ECG monitoring, EchoCG, external respiration function (ERF) test. In both groups, CHD and COPD were treated by the generally accepted standards, Group I patients were additionally given IBB at a water temperature of 37 degrees C; the concentration of iodine and bromine was 10-15 and 30-40 m/I, respectively; the duration was 10-15 min for 2 consecutive days, followed by a rest day or every second day; the course comprised 10-12 sessions. RESULTS: After the course of IBB, there were significant reductions in the number of anginal attacks and the dose of nitrates weekly, the number of episodes of supraventricular and ventricular premature contractions, producing no impact on the cardiac conduction system. The duration of silent myocardial ischemia and the degree of ST-segment depression significantly reduced. No significant changes were obtained in the control group. ERF examination confirmed the safety of IBB used in patients with CHD and COPD. CONCLUSION: IBB has a beneficial antiischemic activity in patients with CHD concurrent with COPD, without producing a negative impact on ERF.

Effects of inhalation of thermal water on exhaled breath condensate in chronic obstructive pulmonary disease.

BACKGROUND: Inhalation of thermal water (TW) is traditionally used as part of the treatment of chronic obstructive pulmonary disease (COPD), but its benefit and mechanisms are controversial. We previously observed a reduced proportion of neutrophils in induced sputum after treatment with TW. OBJECTIVES: The aim of this study was to determine whether inhalation of TW in COPD patients is associated with biochemical changes of airway lining fluid, including a reduction in the neutrophil chemoattractant leukotriene B(4) (LTB(4)). METHODS: Thirteen COPD patients were randomly assigned to receive a 2-week course of TW and normal saline inhalation in a cross-over, single-blind study design. Exhaled breath condensate (EBC) was collected before and after treatments. LTB(4) concentrations in EBC were determined by ELISA, and EBC pH was measured before and after argon deaeration. RESULTS: No significant differences in LTB(4) concentrations in EBC were detected with either treatment. A significant decrease in pH of non-deaerated EBC was observed after a standard course of TW (median 7.45, interquartile range 6.93-7.66, vs. median 6.99, interquartile range 6.57-7.19; p = 0.05), which disappeared after argon deaeration. CONCLUSIONS: There is no evidence that TW treatment affects LTB(4) concentration in EBC. The results of EBC pH measurements suggest that TW inhalation induces an imbalance of volatile components of the buffer system in airway lining fluid.

Use of reverse micellar systems for the extraction and purification of bromelain from pineapple wastes.

Reverse micellar systems of CTAB/isooctane/hexanol/butanol and AOT/isooctane are used for the extraction and primary purification of bromelain from crude aqueous extract of pineapple wastes (core, peel, crown and extended stem). The effect of forward as well as back extraction process parameters on the extraction efficiency, activity recovery and purification fold is studied in detail for the pineapple core extract. The optimized conditions for the extraction from core resulted in forward and back extraction efficiencies of 45% and 62%, respectively, using reverse micellar system of cationic surfactant CTAB. A fairly good activity recovery (106%) and purification (5.2-fold) of bromelain is obtained under these conditions. Reverse micellar extraction from peel, extended stem and crown using CTAB system resulted in purification folds of 2.1, 3.5, and 1.7, respectively. Extraction from extended stem using anionic surfactant AOT in isooctane did not yield good results under the operating conditions employed.

Oxidant-induced cardiomyocyte injury: identification of the cytoprotective effect of a dopamine 1 receptor agonist using a cell-based high-throughput assay.

Myocyte injury due to myocardial reperfusion injury plays a crucial role in the pathogenesis of acute myocardial infarction even after successful coronary revascularization. Identification of compounds that reduce reperfusion-associated myocyte death is important. Therefore, we developed an in vitro model of myocardial reperfusion injury in H9c2 rat cardiomyocytes and applied a cell-based high-throughput approach to screen a standard library of pharmacologically active compounds (LOPAC) in order to identify drugs with cardioprotective effects. Oxidative stress was induced with hydrogen peroxide (H2O2) treatment, which resulted in approximately 50% reduction in cell viability. Test compounds were added at a 3-microM final concentration as a pretreatment or in a delayed fashion (30 min after the peroxide challenge in order to imitate pharmacological treatment following angioplasty). Cells were cultured for 3 or 24 h. Viability was quantitated with the methylthiazolyldiphenyl-tetrazolium bromide method. Cytotoxicity and cytoprotection were also evaluated by measuring the lactate dehydrogenase activity in the cell culture supernatant. The screening identified a number of compounds with cytoprotective action, including molecules that are known to interfere with components of DNA repair and cell cycle progression, e.g. poly(ADP-ribose) polymerase (PARP) inhibitors, topoisomerase inhibitors, and cyclin dependent kinase inhibitors, or reduce energy consumption by interfering with cardiac myofilament function. A number of dopamine D1 receptor agonists also provided significant cytoprotection at 3 h, but only three of them showed a similar effect at 24 h: chloro- and bromo-APB and chloro-PB hydrobromide. Chloro-APB hydrobromide significantly reduced peroxide-induced PARP activation in the myocytes independently of its action on dopamine D1 receptors, but lacked PARP inhibitor capacity in a cell-free PARP assay system. In conclusion, the pattern of cytoprotective drugs identified in the current assay supports the overall validity of our model system. The findings demonstrate that cytoprotective agents, including novel indirect inhibitors of cellular PARP activation can be identified with the method, chloro-APB hydrobromide being one such compound. The current experimental setting can be employed for cell-based high-throughput screening of various compound libraries.

Synthesis and structure-activity relationships of neuromuscular blocking agents.

The first use of neuromuscular blocking agents (muscle relaxants) in clinical practice (1942) revolutionised the practice of anaesthesia and started the modern era of surgery. Since 1942 introduction of tubocurarine (18) neuromuscular blocking agents have been used routinely to provide skeletal muscle relaxation during surgical procedures allowing access to body cavities without hindrance from voluntary or reflex muscle movement. After the introduction of tubocurarine and the depolarizing suxamethonium chloride (4) (1949) several nondepolarizing steroidal and nonsteroidal neuromuscular blocking agents with different onset time and duration of effect were introduced e.g. gallamine triethiodide (1) (1949), methocurine (2) (1949), alcuronium chloride (3) (1963), pancuronium bromide (9) (1968), vecuronium bromide (11) (1982), pipecuronium bromide (10) (1982), atracurium besylate (5) (1982), doxacurium chloride (6) (1991), mivacurium chloride (8) (1992), rocuronium bromide (12) (1994) cisatracurium besylate (7) (1996), and rapacuronium bromide (13) (2000). SZ 1677 (14) a steroid type nondepolarizing neuromuscular blocking agent under development (preclinical phase). This review article deals with a comprehensive survey of the progress in chemical, pharmacological and, in some respects, of clinical studies of neuromuscular blocking agents used in the clinical practice and under development, including the synthesis, structure elucidation, pharmacological actions, structure activity relationships studies of steroidal and nonsteroidal derivatives.

Inactivation of Trypanosoma cruzi dihydrolipoamide dehydrogenase by leukocyte myeloperoxidase systems: role of hypochloride and nitrite related radicals.

Dihydrolipoamide dehydrogenase (LADH) from Trypanosoma cruzi, the causative agent of Chagas' disease, was inactivated by treatment with myeloperoxidase (MPO)-dependent systems. LADH lipoamide reductase and diaphorase activities decreased as a function of incubation time and composition of the MPO/H2O2/halide system, a transient increase preceding the loss of diaphorase activity. Iodide, bromide, thiocyanide and chloride were effective components of MPO/H2O2 or MPO/NADH systems. Catalase prevented LADH inactivation by the MPO/NADH/halide systems in agreement with H2O2 production by NADH-supplemented LADH. Thiol compounds (L-cysteine, N-acetylcysteine, penicillamine, N-(2-mercaptopropionylglycine) and Captopril prevented LADH inactivation by the MPO/H2O2/NaCl system and by NaOCl, thus supporting HOCl as agent of the MPO/H2O2/NaCl system. MPO/H2O2/NaNO2 and MPO/NADH/NaNO2 inactivated LADH, the reaction being prevented by MPO inhibitors and thiol compounds. T. cruzi LADH was affected by MPO-dependent systems like myocardial LADH, allowance being made for the variation of the diaphorase activity and the greater sensitivity of the T. cruzi enzyme to MPO/H2O2/halide systems.

Distribution and ontogeny of 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid-sensitive and quisqualate-insensitive [3H]glutamate binding sites in the rat brain.

Displacement of [3H]glutamate by 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid and quisqualate (in the presence of saturating concentrations of ionotropic glutamate receptor agonists) was used to characterize optimal ionic conditions, distribution, and the ontogeny of glutamate receptor binding sites in rat brain. Using rat forebrain membranes or receptor autoradiography, optimal 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid-sensitive [3H]glutamate binding was found in the presence of 100 mM bromide ions and in the absence of calcium ions. Under these conditions, [3H]glutamate binding was relatively quisqualate insensitive. In regions of the neonatal (11-day-old) and adult rat brain, this [3H]glutamate binding was highest in forebrain (striatum, cerebral cortex, and hippocampus) and hypothalamus/mid-brain but was lower in the cerebellum, olfactory bulb, and pons/medulla regions. 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid-sensitive and quisqualate-insensitive [3H]glutamate binding was present in the rat forebrain at 1 day of age and gradually increased more than twofold by day 50 (adult). Thus, in the presence of bromide ions and in the absence of calcium ions, [3H]glutamate labels a subpopulation of metabotropic glutamate receptors that are sensitive to 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid but insensitive to quisqualate. Expression of [3H]glutamate binding under these conditions was both regionally and developmentally regulated in rat brain, suggesting that [3H]glutamate is labeling a distinct population of metabotropic glutamate receptors.

Characterization of the bifunctional cytochrome c reductase-processing peptidase complex from potato mitochondria.

In potato, cytochrome c reductase, a protein complex of the respiratory chain, exhibits processing activity toward mitochondrial precursor proteins. One of the two cooperating components of the processing peptidase was shown to be identical with subunit III of the complex. Here we report that two additional proteins of the complex (subunit I and II) share 40-50% sequence identity with the processing enhancing protein, the other component of the processing enzyme from fungi and mammals. Thus the composition and structure of the complex integrated processing peptidase seems to be different from its fungal and mammalian counterparts. Cytochrome c reductase from potato is extraordinarily stable, and separation of subunit III from the complex leads to aggregation of the remaining subcomplex and irreversible loss of processing activity. Expression of the three high molecular weight subunits of the complex allowed purification of each individual protein. Neither the individual subunits nor their combinations are active in in vitro processing assays suggesting that they may need the structural support of the complex for activity. In contrast to mitochondrial processing peptidases from other organisms, the purified potato enzyme is active in the presence of high salt (above 1 M NaCl) and works efficiently without addition of metal ions. These data indicate that potato cytochrome c reductase is a bifunctional protein complex with unique features. Possibly, there is a more general evolutionary relationship between cytochrome c reductases and mitochondrial processing peptidases than hitherto assumed.

Dietary fluoride, unlike bromide or iodide, counteracts phosphorus-induced nephrocalcinosis in female rats.

Supplemental dietary F has been shown to counteract P-induced nephrocalcinosis in female rats. In order to obtain information as to the specificity of this F effect, the effect of other halogens, namely Br and I, on P-induced nephrocalcinosis was studied in weanling female rats. Supplemental dietary Br (5.24 mmol/kg of diet) and I (1.43 mmol/kg of diet) did not influence P-induced nephrocalcinosis, whereas F at equimolar dietary concentrations had marked antinephrocalcinogenic activity. The halogens were added to the diets in the form of KBr, KI, and NaF; the diets were balanced for the kations with Cl salts. The addition of KI to the diet to a concentration of 5.24 mmol/kg caused pronounced growth retardation, decreased feed intake, hepatomegaly, and signs of lethargy. It is concluded that the protective effect of dietary F against P-induced nephrocalcinosis does not extend to other halogens.

Inactivation of DNA-mediated transformation of hamster cells by gamma-rays and deoxyribonuclease I.

DNA damage has been induced in the mammalian expression vector pSV2-gpt by irradiation with X-rays or treatment with deoxyribonuclease I (DNAase I) under controlled conditions in vitro. The biological effect of such treatment was assessed by stable gene expression in Chinese hamster ovary (CHO) cells using DNA-mediated gene transfer. Induction of DNA double-strand breaks (dsbs), resulting from the interaction of independently-induced single-strand breaks (ssbs) under the present conditions, was measured by agarose gel electrophoresis of the treated vector. The correlation between radiation-induced gene inactivation and dsb induction mediated by OH radicals suggests that a dsb in the gene is a major inactivating lesion in this system. Individual radiation-induced ssbs and nucleotide damage are produced much more frequently than dsbs under these conditions, but the majority of these lesions do not appear to inactivate the gpt gene. DNAase I treatment, giving only simple 5' P + 3' OH breaks in the vector DNA, gave a correlation of approximately 1.5 dsb in the gpt gene per inactivating event, confirming little repair of dsbs in this system. Inactivation of the gpt gene without appreciable formation of dsbs was found, however, when the vector was irradiated at high dose rate in the presence of the OH-radical scavenger KBr. The nature of non-break damage causing inactivation requires further study.