CCS5, a Thioredoxin-like Protein Involved in the Assembly of Plastid c-Type Cytochromes

The c-type cytochromes are metalloproteins with a heme molecule covalently linked to the sulfhydryls of a CXXCH heme-binding site. In plastids, at least six assembly factors are required for heme attachment to the apo-forms of cytochrome f and cytochrome c₆ in the thylakoid lumen. CCS5, controlling plastid cytochrome c assembly, was identified through insertional mutagenesis in the unicellular green alga Chlamydomonas reinhardtii. The complementing gene encodes a protein with similarity to Arabidopsis thaliana HCF164, which is a thylakoid membrane-anchored protein with a lumen-facing thioredoxin-like domain. HCF164 is required for cytochrome b₆f biogenesis, but its activity and site of action in the assembly process has so far remained undeciphered. We show that CCS5 is a component of a trans-thylakoid redox pathway and operates by reducing the CXXCH heme-binding site of apocytochrome c prior to the heme ligation reaction. The proposal is based on the following findings: 1) the ccs5 mutant is rescued by exogenous thiols; 2) CCS5 interacts with apocytochrome f and c₆ in a yeast two-hybrid assay; and 3) recombinant CCS5 is able to reduce a disulfide in the CXXCH heme-binding site of apocytochrome f.     

Effects of the cholesteryl ester transfer protein inhibitor torcetrapib on VLDL apolipoprotein E metabolism

Cholesteryl ester transfer protein (CETP) inhibition leads to changes in lipoprotein metabolism. We studied the effect of the CETP inhibitor torcetrapib on VLDL apolipoprotein E (apoE) metabolism. Subjects, pretreated with atorvastatin (n = 9) or untreated (n = 10), received placebo followed by torcetrapib (4 weeks each). After each treatment, subjects underwent a primed-constant infusion of D(3)-leucine to determine the VLDL apoE production rate (PR) and fractional catabolic rate (FCR). Torcetrapib alone reduced the VLDL apoE pool size (PS) (-28%) by increasing the VLDL apoE FCR (77%) and leaving the VLDL apoE PR unchanged. In subjects pretreated with atorvastatin, torcetrapib increased the VLDL apoE FCR (25%) and PR (21%). This left the VLDL apoE PS unchanged but increased the VLDL apoE content, likely enhancing VLDL clearance and reducing LDL production in this group. Used alone, torcetrapib reduces the VLDL apoE PS by increasing the apoE FCR while leaving the VLDL apoE content unchanged. In contrast, torcetrapib added to atorvastatin treatment increases both the VLDL apoE FCR and PR, leaving the VLDL apoE PS unchanged. Adding torcetrapib to atorvastatin treatment increases the VLDL apoE content, likely leading to decreased conversion of VLDL to LDL, reduced LDL production, and lower levels of circulating VLDL and LDL.     

A new species of Dyrosaurus (Crocodylomorpha, Dyrosauridae) from the early Eocene of Morocco: phylogenetic implications

A new dyrosaurid is described from the Ypresian of the phosphatic deposits of the Oulad Abdoun Basin of Morocco. It is based on numerous cranial and postcranial remains, allowing an almost complete reconstruction. This new Dyrosaurus species, Dyrosaurus maghribensis sp. nov. , is currently only known from Morocco. It differs from D. phosphaticus , present in contemporaneous levels of Algeria and Tunisia, by several autapomorpies, including a smooth dorsal margin of the parietal and widely opened choanae. A phylogenetic analysis, using 47 taxa and 234 morphological characters, shows the dyrosaurids as the sister taxon of pholidosaurids, which include Elosuchus , Sarcosuchus , Terminonaris and Pholidosaurus , and the thalattosuchians. Goniopholididae is a non-monophyletic group; however, if dyrosaurids are not included in the analysis, the result differs and the goniopholidids form a distinct clade. If Thalattosuchia is excluded, both Goniopholididae and Pholidosauridae become paraphyletic assemblages. Thus, phylogenetic problems remain with respect to longirostrine clade, and more attention should be paid to resolving their evolutionary relationships amongst the crocodyliforms.  © 2006 The Linnean Society of London, Zoological Journal of the Linnean Society , 2006, 148 , 603–656.     

Isolation and characterization of a new hemoglobin derivative cross-linked between the alpha chains (lysine 99 alpha 1----lysine 99 alpha 2)

Bis(3,5-dibromosalicyl) fumarate and a number of related bifunctional reagents react preferentially with oxyhemoglobin to cross-link the beta chains within the 2,3-diphosphoglycerate-binding site. In this report we describe a new derivative cross-linked between the alpha chains which is formed specifically in the reaction with deoxyhemoglobin. X-ray crystallographic studies show that the cross-link lies between Lys-99 alpha 1 and Lys-99 alpha 2, spanning the central cavity of the tetramer. Lys-99 alpha 1 and Lys-99 alpha 2 are located within a cluster of charged residues very near the middle of the hemoglobin molecule. In oxyhemoglobin, this site is completely inaccessible to the cross-linking agent. Competition experiments with inositol hexaphosphate indicate that the compound enters the central cavity in deoxyhemoglobin through the cleft between the alpha chains. Despite the presence of the cross-link between the alpha chains, the modified hemoglobin remains highly cooperative. The Hill coefficient for HbXL99 alpha is 2.6. The oxygen affinity of the cross-linked derivative is decreased by approximately 2-fold; at pH 7.0 in the presence of 0.1 M NaCl the P50 is 13.9 mm Hg compared to 6.6 mm Hg for HbA. This difference appears to be due to relatively small changes in both KR, the association constant for binding of oxygen to the R state, and the allosteric constant L. Surprisingly, the isoelectric point of oxyHbXL99 alpha is almost identical to that of oxyHbA, whereas in the deoxy form the isoelectric point of the cross-linked derivative is decreased relative to native hemoglobin as expected due to the loss of the two positive charges of the modified amino groups. In agreement with these findings, the alkaline Bohr effect of HbXL99 alpha is decreased by more than 50%. Earlier studies argue strongly against the possibility that Lys-99 alpha is directly responsible for this large fraction of the Bohr effect in HbA. Analysis of the structure suggests that in the cross-linked derivative Glu-101 beta, which is in close proximity to Lys-99 alpha in oxyhemoglobin, becomes an acid Bohr group.     

Taurine stimulation of isolated hamster brain Na+,K+-ATPase: activation kinetics and chemical specificity

Epileptic foci are associated with locally reduced taurine (2-aminoethanesulfonic acid) concentration and Na+,K+-ATPase (EC 3.6.1.3) specific activity. Topically applied and intraperitoneally administered taurine can prevent the development and/or spread of foci in many animal models. Taurine has been implicated as a possible cytosolic modulator of monovalent ion distribution, cytosolic "free" calcium activity, and neuronal excitability. Taurine may act in part by modulating Na+,K+-ATPase activity of neuronal and glial cells. We characterized the requirements for in vitro modulation of Na+,K+-ATPase by taurine. Normal whole brain homogenate Na+,K+-ATPase activity is 5.1 +/- 0.4 (4) mumol Pi X h-1 X mg-1 Lowry protein. Partial purification of the plasma membrane fraction to remove cytosolic proteins and extrinsic proteins and to uncouple cholinergic receptors yields a membrane-bound Na+,K+-ATPase activity of 204.6 +/- 5.8 (4) mol Pi X h-1 X mg-1 Lowry protein. Taurine activates the Na+,K+-ATPase at all levels of purification. The concentration dependence of activation follows normal saturation kinetics (K1/2 = 39 mM taurine, activation maximum = +87%). The activation exhibits chemical specificity among the taurine analogues and metabolites: taurine = isethionic acid greater than hypotaurine greater than no activation = beta-alanine = methionine = choline = leucine. Taurine can act as an endogenous activator/modulator of Na+,K+-ATPase. Its action is mediated by a membrane-bound protein.     

Mouse Toxicity of Fungi of Tobacco

A bioassay for fungal toxins based on the intraperitoneal injection of test materials into mice was used to screen 976 cultures isolated from tobacco and grown in a high-protein baby cereal and also to determine whether samples of tobacco damaged by fungi are more toxic than samples of apparently sound tobacco. Of 236 fungal isolates from noncured tobacco, 79% were lethal when homogenized cultures of these isolates were tested. Forty-nine per cent of 740 fungi isolated from cured tobacco were lethal. Of the genera from which 30 or more isolates were tested, Epicoccum, Alternaria, and Penicillium had the highest percentage of toxic isolates from non-cured tobacco, whereas Epicoccum, Aspergillus, and Alternaria had the highest percentage from cured tobacco. Samples of tobacco naturally infected with brown spot, caused by Alternaria tenuis, did not have a significantly different LD(50) value after 48 hr than comparable disease-free samples. However, animals which died from doses near the LD(50) dose of tobacco infected with Alternaria generally died in 24 to 48 hr with signs associated with a depressant rather than a stimulant, such as nicotine, which caused death in 15 to 30 min. These signs were duplicated by injecting homogenized pure cultures of Alternaria. These studies, although inconclusive with regard to the effects of fungal contaminants on the quality or usability of tobacco, have developed evidence that suggests the advisability of a study on smoke or smoke condensates from moldy and nonmoldy tobacco.     

Fungi Isolated from Flue-cured Tobacco at Time of Sale and After Storage

The fungi isolated from 100 samples of flue-cured tobacco from 12 markets in 2 tobacco belts comprised 11 genera, including 10 species of Aspergillus. The mean percentage per sample isolated from 62 samples of tobacco from Middle Belt markets was Alternaria, 40.6%; Aspergillus niger, 47.8%; Aspergillus repens, 38.0%; and Penicillium, 25.8%. The mean percentage per sample isolated from 38 samples of tobacco from Old Belt markets was Alternaria, 74.0%; Penicillium, 52.5%; Aspergillus repens, 38.0%; and Aspergillus ruber, 36.2%. Damaged (74 samples) and nondamaged (26 samples) stored tobacco yielded species of six genera of fungi, including eight species of Aspergillus. Species of Aspergillus and Penicillium were commonly isolated from both damaged and nondamaged tobacco, whereas species of Alternaria, Cladosporium, Fusarium, and Rhizopus were isoalted more frequently from nondamaged tobacco. The fungi that occurred in the highest population in damaged tobacco were Aspergillus repens, A. niger, A. ruber, and Penicillium species.     

Fungi Isolated from Flue-Cured Tobacco Sold in Southeast United States, 1968-1970

Flue-cured tobacco leaves, from low- and middle-stalk positions, offered for sale in each of two markets, within each of five tobacco types, were evaluated for moisture content (MC) and filamentous fungi during August through October in 1968, 1969, and 1970. Alternaria alternata, Penicillium cyclopium, Aspergillus niger, Aspergillus repens, and Aspergillus flavus were most frequently isolated from cultured tissue. Other filamentous fungi that grew from the tissue included species from four genera of field fungi and seven species of storage fungi. Although the MC ranged from 11.0 to 22.5%, it averaged 16.4, 16.8, and 15.9% for samples taken in 1968, 1969, and 1970, respectively. Average populations of fungi per sample over the three years ranged from 0 to 1,528,500 colonies/g of tobacco.