Pharmacological characterization of the effects of taurine on calcium uptake in the rat retina

Summary Taurine is known to increase ATP-dependent calcium ion (Ca²⁺) uptake in retinal membrane preparations and in isolated rod outer segments (ROS) under low calcium conditions (10M) (Pasantes-Morales and Ordóñez, 1982; Lombardini, 1991). In this report, ATP-dependent Ca²⁺ uptake in retinal membrane preparations was found to be inhibited by 5M cadmium (Cd²⁺), suggesting the involvement of cation channel activation. The activation of cGMP-gated cation channels, which are found in the ROS, is a crucial step in the phototransduction process. An inhibitor of cGMP-gated channels, LY83583, was found to inhibit taurine-stimulated ATP-dependent Ca²⁺ uptake but had no effect on ATP-dependent Ca²⁺ uptake in the absence of taurine, indicating that taurine may be increasing ATP-dependent Ca²⁺ uptake through a mechanism of action involving the opening of cGMP-gated channels. The activation of cGMP-gated channels with dibutyryl-cGMP and with phosphodiesterase inhibition using zaprinast caused an increase in ATP-dependent Ca²⁺ uptake in isolated ROS, but not in taurine-stimulated ATP-dependent Ca²⁺ uptake. LY83583 had the same effects in isolated ROS as in retinal membrane preparations. Another inhibitor of cGMP-gated channels, Rp-8-Br-PET-cGMPS, produced the same pattern of inhibition in isolated ROS as LY83583. Thus, there appears to be a causal link between taurine and the activation of the cGMP-gated channels in the ROS under conditions of low calcium concentration, a connection that suggests an important role for taurine in the visual signalling function of the retina.     

Synthesis and characterization of the asymmetrically coordinated dinuclear complex [{L(Ph2acac)FeIII}(μ-O){FeIII(Cl4-cat)L}](ClO4) and its one-electron reduced and oxidized forms. Models for dinuclear non-heme active sites

The asymmetrically coordinated complex [{L(Ph₂acac)Fe^III}(μ-O){Fe^III(Cl₄-cat)L}](BPh₄)·1.5toluene has been synthesized and structurally characterized (Ph₂acac^–=1,3-diphenylpropane-1,3-dionate, Cl₄-cat^2–=tetrachlorocatecholate, L=1,4,7-trimethyl-1,4,7-triazacyclononane). This species can be electrochemically oxidized and reduced by one electron, respectively, yielding two species which both have an S=1/2 ground state. It is shown that the oxidation is ligand-centered, affording a coordinated semiquinonate(1–) ligand with S=1/2 which is antiferromagnetically coupled to a high-spin Fe^III ion (S=5/2) yielding an S=2 state which, in turn, is antiferromagnetically coupled (through the oxo bridge) to the second high-spin Fe^III ion (S=5/2) yielding the observed S=1/2 ground state. In contrast, the reduction is metal-centered generating a mixed-valent species with an [Fe^III-O-Fe^II]³⁺ core; intramolecular antiferromagnetic coupling again produces an S=1/2 ground state. The symmetrical complex [{LFe^III(Ph₂acac)}₂(μ-O)](ClO₄)₂ has also been synthesized, as have the mononuclear species [LFe^II(Ph₂acac)Cl] and [LFe^III(aacac)Cl](ClO₄)·1 mesitylene [aacac^–=3-(9-anthryl)acetylacetonate(1–)], all of which have been characterized by X-ray crystallography. The magnetism, the Mössbauer-, EPR-, and UV-VIS-spectra and the electrochemistry of complexes are reported.     

Metal binding to the tetrathiolate motif of desulforedoxin and related polypeptides

 Desulforedoxin and the N-terminus of desulfoferrodoxin share a 36 amino acid domain containing a (Cys-S)₄ metal binding site. Recombinant forms of desulforedoxin, an N-terminal fragment of desulfoferrodoxin, and two desulforedoxin mutant proteins were reconstituted with Fe³⁺, Cd²⁺, and Zn²⁺ and relative metal ion affinities assessed by proton titrations. Protons compete with metal for protein ligands, a process that can be followed by monitoring the optical spectrum of the metal-protein complex as a function of pH. For all polypeptides, Fe³⁺ bound with the highest affinity, whereas the affinity of Zn²⁺ was greater than Cd²⁺ in desulforedoxin and the N-terminal fragment of desulfoferrodoxin, but this order was reversed in desulforedoxin mutant proteins. Metal binding in both mutants was significantly impaired. Furthermore, the Fe³⁺ complex of both mutants underwent a time-dependent bleaching process which coincided with increased reactivity of cysteine residues to Ellman's reagent and concomitant metal dissociation. It is hypothesized that this results from an autoredox reaction in which Fe³⁺ is reduced to Fe²⁺ with attendant oxidation of ligand thiols. Received: 17 June 1998 / Accepted: 3 September 1998     

Formation and movement of Fe(III) in horse spleen, H- and L-recombinant ferritins. A fluorescence study.

Iron oxidation and incorporation into apoferritins of different subunit composition, namely the recombinant H and L homopolymers and the natural horse spleen heteropolymer (10-15% H), have been followed by steady-state and time-resolved fluorescence. After aerobic addition of 100 Fe(II) atoms/polymer, markedly different kinetic profiles are observed. In the rL-homopolymer a slow monotonic fluorescence quenching is observed which reflects binding, slow oxidation at the threefold apoferritin channels, and diffusion into the protein cavity. In the rH-homopolymer a fast fluorescence quenching is followed by a partial, slow recovery. The two processes have been attributed to Fe(II) binding and oxidation at the ferroxidase centers and to Fe(III) released into the cavity, respectively. The fluorescence kinetics of horse spleen apoferritin is dominated by the H chain contribution and resembles that of the H homopolymer. It brings out clearly that the rate of the overall process is limited by the rate at which Fe(III) leaves the ferroxidase centers of the H chains where binding of incoming Fe(II) and its oxidation take place. The data obtained upon stepwise addition of iron and the results of optical absorption measurements confirm this picture. The correspondence between steady-state and time-resolved data is remarkably good; this is manifest when the latter are used to calculate the change in fluorescence intensity as apparent in the steady-state measurements.     

Iron accumulation in tobacco plants expressing soyabean ferritin gene

High iron-content transgenic tobacco plants have been produced by transfer via Agrobacterium tumefaciens of soyabean ferritin cDNA under the control of a CaMV 35S promoter. Immunoblot analysis of protein from transgenic tobacco plants suggested mature ferritin subunits are produced by cleavage of transit peptides. The expressed ferritin was observed in the tissues of leaves and stems. The maximal iron content of transformant leaves was approximately 30% higher than leaves from non-transformants. The increased iron content of each transformant was correlated with increases in ferritin content. These results demonstrate the potential of breeding high iron content crops by introduction of the ferritin gene     

用膨胀床金属亲和层析从淡菜匀浆液中分离纯化纤维素酶

研究了一种新的膨胀床金属亲和层析技术,即将金属亲和层析结合膨胀床层析,直接从淡菜(Blue mussel)匀浆液中纯化纤维素酶。研究了金属亲和配基种类、pH、离子强度及流速对酶吸附和解吸的影响,确定了酶洗脱条件和介质再生条件。一步可纯化纤维素酶194倍,酶收率达82%。本方法不需要预先去除细胞碎片,而且处理速率比传统层析技术高3～4倍。     

杀灭土壤中线虫对小麦生长和吸收N、P的影响

盆栽条件下研究了施用杀线剂(克线磷,67mg·kg^-1干土)和干热(105℃,2h)两种杀线措施对小麦生长和N、P养分吸收的影响。杀线剂对土壤中线虫的平均杀灭率约为80%, 干热处理的杀灭率为100%. 在杀线剂处理中,苗期至抽穗期小麦生物量、拔节期至成熟期植株含N量、全生育期植株吸N量以及抽穗和成熟期吸P量均显着低于对照。土壤干热处理后抽穗和成熟期小麦的生物量、含N量及N、P吸收量也比对照显着降低。两种杀线处理植株地上部生物量和N、P吸收量与相应处理全株变化趋势基本一致。但杀灭线虫对植株含P量影响较小。分析杀线虫后小麦生长和养分吸收受抑主要与土壤有机氮的矿化作用减弱、微生物活动产生的植物生长促进物质降低有关     

Iron Binding and Autoreduction by Citrate: Are These Involved in Signalling by Iron Regulatory Protein-l?

Ferric ions bind to citrate and undergo an autoreduction to form a ferrous-citrate complex, greatly increasing the redox activity of the iron complex. Ferrous ions and citrate are also essential for the enzymic activity of aconitase. Aconitase, with its iron-sulphur cluster has a versatile structure which allows it to act as an iron regulatory protein (IW-1). The purpose of this study was to see whether iron binding, and its autoreduction by citrate, could play a physiological signalling role in iron regulation. Significant amounts of ferrous ions were associated with citrate, when measured using ferrozine, however, these did not appear to activate iron-requiring aconitase.     

A Marked Increase in Free Copper Levels in the Plasma and Liver of LEC Rats: An Animal Model for Wilson Disease and Liver Cancer

Most of copper present in rat plasma and liver binds to caeruloplasmin and metallothionein, respectively, and is not redox active. However, free forms of copper including loosely bound forms to other molecules are redox active. We assessed the free copper in Long-Evans rats with a cinnamon-like coat color (LEC rats), an animal model of Wilson disease and liver cancer. Compared to those of control rats, the liver and plasma of LEC rats showed a marked elevation of free copper, especially at the stage of acute hepatitis, in parallel with an increase of total copper levels in the livers and a decrease of plasma caeruloplasmin (ferroxidase I) activity. At the onset of jaundice, the total copper levels, however, decreased in liver, but increased in plasma, while free copper levels in both liver and plasma remained higher. Free iron levels in both liver and plasma were also determined and did not change significantly, except for the case of plasma in jaundiced rats. The data are consistent with a proposal in which increased levels of redox active free copper in the liver of LEC rats catalyze Fenton-type reactions, producing a large flux of hydroxyl radicals that would play an important role in the observed liver dysfunction, leading to acute hepatitis, and, finally, hepatocarcinoma. This is the first demonstration that the free copper may participate in the pathophysiology of the LEC rats and Wilson disease.     

The influence of aquatic surface respiration (ASR) on cardio-respiratory function of the serrasalmid fish Piaractus mesopotamicus

When exposed to severely hypoxic water, many teleosts skim the better oxygenated surface layer (aquatic surface respiration, ASR). Information is scarce concerning the thresholds triggering ASR and its cardio-respiratory consequences. To assess the ambient conditions leading to ASR and to evaluate its effects on cardio-respiratory function, we exposed specimens of Piaractus mesopotamicus to gradual hypoxia (water oxygen tension ranging from 120 to 10 torr) with or, alternatively, without access to the surface. Concurrently, ASR, cardiac and respiratory frequencies, O₂ uptake and gill ventilation were monitored. With surface access, ASR developed below the critical tension for O₂ uptake (34 torr) by normal gill ventilation. Moreover, the time spent in ASR increased with prolonged hypoxic exposure to a maximum of 95% of total time. Without surface access, the species exhibited hypoxic bradycardia, that had not occurred in the group with fully developed ASR. Even without ASR, P. mesopotamicus recovered readily from hypoxic exposure, showing that this species possesses a number of mechanisms to cope with environmental hypoxia.