A comparative study of protoheme and heme d catalases : role of the heme and the heme pocket in catalysis and ligand binding

Catalase dismutes H20 2 to O2 and H20. In successive twoelectron reactions H20 2 induces both oxidation and reduction at the heme group. In the first step the protoheme prosthetic group of beef liver catalase forms compound I, in which the heme has been oxidized from Fe3+ to Fe4+=0 and a porphyrin radical has been created. Compound II is formed by the oneelectron reduction of comp I. It retains Fe4+=0 but lacks the porphyrin radical and is catalytically inert. Molecular structures are available for Escherichia coli Hydroperoxidase II, Micrococcus Iysodeiktus, Penicillium vitale and beef liver enzymes, which contain different hemes and heme pockets. In the present work, the pockets and substrate access channels of protoheme (beef liver & Micrococcus) and heme d (HPII of E. coli and Penicillium) catalases have been analysed using Quanta™ and CharmMTM molecular modeling packages on the Silicon Graphics Iris Indigo 2 computer. Experimental studies have been carried out with two catalases, HPII (and its mutants) and beef liver. Fluoride and formate' are inhibitors of both enzymes, and their binding is modulated by the heme and by distal residues N201 & H128. Both HPII and beef liver enzymes form compound I with H202 or peracetate. The reduction of beef liver enzyme compound I to II and the decay of compound II are accelerated by fluoride. The decay of compound II is also accelerated by formate, and this reagent acts as a 2-electron donor towards compound I of both enzymes. It is concluded that heme d enzymes (Penicillium and HPII of E. coli) are formed by autocatalytic transformation of protoheme in a modified pocket which contains a characteristic serine residue as well as a partially occluded heme channel. They are less active than protoheme enzymes but also do not form the inactive compound II species. Binding of peroxide as well as fluoride and formate is prevented by mutation of H128 and modulated by mutation of N201.

Cloning of actin genes from a genomic library of the newt, Notophthalmus viridescens

The regenerating urodele limb is a useful model system in which to study, in vivo, the controls of cell proliferation and differentiation. Techniques are available which enable one to experimentally manipulate mitogenic influences upon the blastema, as well the morphogenesis of the regenerating 11mb. Although classical regeneration studies have generated a wealth of knowledge concerning tissue interactions, little 1s known about the process at the level of gene expression. The aim of this project was to clone potentially developmentally regulated genes from a newt genomic library for use in future studies of gene expression during limb regeneration. We decided to clone the cytoskeletal actin gene for the following reasons: 1. its expression reflects the proliferative and differentiatlve states of cells in other systems 2. the high copy number of cytoplasmic actin pseudogenes in other vertebrates and the high degree of evolutionary sequence conservation among actin genes increased the chance of cloning one of the newt cytoplasmic actin genes. 3. Preliminary experiments indicated that a newt actin could probably be identified using an available chick ~-actln gene for a molecular probe. Two independent recombinant phage clones, containing actin homologous inserts, were isolated from a newt genomic library by hybridization with the chick actin probe. Restriction mapping identified actin homologous sequences within the newt DNA inserts which were subcloned into the plasmid pTZ19R. The recombinant plasmids were transformed into the Escherichia coli strain, DHsa. Detailed restriction maps were produced of the 5.7Kb and 3.1Kb newt DNA inserts in the plasmids, designated pTNAl and pTNA2. The short «1.3 Kb) length of the actin homologous sequence in pTNA2 indicated that it was possibly a reverse transcript pseudogene. Problems associated with molecular cloning of DNA sequences from N. viridescens are discussed with respect to the large genome size and abundant highly repetitive DNA sequences.

Palaeopigment accumulation and implcations for paleolimnology over the lost century for two Ontario lakes

Crawford Lake is a meromictic lake, which is 24 m deep and has an area of 2.5 ha, and has never been reported to have mixed below 16 m. Lady Evelyn Lake, which became a reservoir when a dam was built in 1916, is dimictic with a maximum depth of about 35 m. 1 My research proved that both native chlorophylls and the ratio of chlorophyll derivatives to total carotenoids were better preserved in the shallower lake (Crawford Lake) because it was meromictic. Thus the anaerobic conditions in Crawford Lake below 16 m (monimolimnion) provide excellent conditions for pigment preservation. Under such conditions, the preservation of both chlorophylls and carotenoids, including oscillaxanthin and myxoxanthophyll, are extremely good compared with those of Lady Evelyn Reservoir, in which anaerobic conditions are rarely encountered at the mud-water interface. During the period from 1500 to 1900 A. D. in Crawford Lake, the accumulation rates of oscillaxanthin and myxoxanthophyll were extremely high, but those of chlorophyll derivatives and total carotenoids were relatively low. This was correlated with the presence of a dense benthic mat of cyanobacteria near the lake's chemocline. Competition for light between the deep dwelling cyanobacteria and overlying phytoplankton in this meromictic lake would have been intensified as the lake became more and more eutrophic (1955-1991 A. D.). During the period from 1955 to 1991 A. D., the accumulation rates of chlorophyll derivatives and total carotenoids in the sediment core from Crawford Lake (0-7.5 cm, 1955-present) increased. During this same period, the accumulation rates of cyanobacterial pigments (Le. oscillaxanthin and myxoxanthophyll) declined as the lake became more eutrophic. Because the major cyanobacteria in Crawford Lake are benthic mat forming Lyngbya and Oscillatoria and not phytoplankton, eutrophication resulted in a decline of the mat forming algal pigments. This is important because in previous palaeolimnological studies the concentrations of oscillaxanthin and myxoxanthophyll have been used as correlates with lake trophic levels. The results of organic carbon a13c analysis on the Crawford Lake sediment core supported the conclusions from the pigment study as noted above. High values of a13c at the depth of 34-48 cm (1500-1760 A. D.) were related to a dense population of benthic Oscillatoria and Lyngbya living on the bottom of the lake during that period. The Oscillatoria and Lyngbya utilized the bicarbonate, which had a high a 13C value. Very low values were found at 0-7 cm in the Crawford sediment core. At this time phytoplankton was the main primary producer, which enriched 12C by photosynthetic assimilation.

Paleoindicators of meromixis

This study was undertaken to ascertain whether meromictic lakes could be differentiated from holomictic lakes on the basis of their surficial profundal sediments. Surface sediment cores (15 cm long) were collected from both the littoral and profundal zones of four meromictic and six holomictic lakes and analyzed for total number of fossil chironomid headcapsu~es, chlorophyll and carotenoid degradation products as well as \ iron and manganese concentrations. Littoral and profundal comparisons of the surface sediments were made between the two lake types using the Mann-Whitney U test. Iron, manganese and the iron to manganese ratio in the littoral sediments of meromictic lakes were significantly lower than those found in the littoral sediments of holomictic lakes. The observed differences are believed to represent an artifact of the significantly higher carbonate concentrations found in three of the four meromictic lakes studied. Profundal and littoral to profundal ratio comparison between holomictic and meromictic lakes suggest that the significantly lower iron and higher carotenoid concentrations in meromictic profundal sediments were a con~equence of meromixis. However, the overlap in distribution exhibited by both iron and carotenoid degradation products between the two lake types was sufficiently large in this study to nullify their use as a means of differentiating meromictic from holomictic lakes. A long core (4.25 m) was removed from the deepest part of the meromictic Crawford Lake (Ontario), sectioned at 5 cm intervals, and analyzed to assess when meromixis occurred, based on its fossil record. Temporal changes in the total number of chironomid headcapsules, and chlorophyll and carotenoid sediment degradation products were closely correlated with organic matter, indicating in my opinion that extensive redeposition of littoral chironomid headcapsules in the profundal zone has occurred. Temporal variations in carotenoid degradation products, in response to changes in organic matter, obscured increased preservation that may have occurred as a consequence of meromixis. Temporal variations in iron and manganese suggest that relatively stable redox conditions have existed throughout most of the lake's history. Therefore it would appear that Crawford Lake has been meromictic since its inception.

Characterization of the state transition in the cyanobacterium synechococcus sp. 7002 and a phycobilisome-less Mutant

ABSTRACT Photosynthetic state transitions were investigated in the cyanobacterium Synechococcus sp. PCC 7002 in both wild-type cells and mutant cells lacking phycobilisomes. Preillumination in the presence of DCMU (3(3,4 dichlorophenyl) 1,1 dimethyl urea) induced state 1 and dark adaptation induced state 2 in both wild-type and mutant cells as determined by 77K fluorescence emission spectroscopy. Light-induced transitions were observed in the wildtype after preferential excitation of phycocyanin (state 2) or preferential excitation of chlorophyll .a. (state 1). The state 1 and 2 transitions in the wild-type had half-times of approximately 10 seconds. Cytochrome f and P-700 oxidation kinetics could not be correlated with any current state transition model as cells in state 1 showed faster oxidation kinetics regardless of excitation wavelength. Light-induced transitions were also observed in the phycobilisomeless mutant after preferential excitation of short wavelength chlorophyll !l. (state 2) or carotenoids and long wavelength chlorophyll it (state 1). One-dimensional electrophoresis revealed no significant differences in phosphorylation patterns of resolved proteins between wild-type cells in state 1 and state 2. It is concluded that the mechanism of the light state transition in cyanobacteria does not require the presence of the phycobilisome. The results contradict proposed models for the state transition which require an active role for the phycobilisome.

Mechanisms underlying Retinoic acid-induced chemoattraction in molluscan neurons

Retinoic acid, a derivative of vitamin A, is known to play diverse roles in development and regeneration. Previous research in the mollusc Lymnaea stagnalis has shown that a gradient of all-trans retinoic acid attracts the growth cones of cultured neurons. The present study investigates the sub-cellular mechanisms within the growth cones of Lymnaea pedal A neurons which mediate the attractive response to a gradient of alltrans retinoic acid. In this study, the mechanism of growth cone turning is shown to be local, as neurites mechanically isolated from their cell body retain the capacity to turn towards an exogenous gradient of all-trans retinoic acid. The turning response is dependent on the initiation of protein synthesis and calcium influx, but does not appear to involve signaling through protein kinase C (PKC). The retinoid X receptor (RXR), which classically functions as a transcription factor, was also shown to be involved in the turning response, functioning locally through a non-genomic pathway. These data show, for the first time in any species, that all-trans retinoic acid's chemotropic action involves a local mechanism involving non-genomic signaling through the RXR. As retinoic acid is known to playa role in regeneration, understanding the mechanisms underlying retinoic acid signaling may lead to further advances in regenerative neuroscience.