A novel and remarkably thermostable ferredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus.

The archaebacterium Pyrococcus furiosus is a strict anaerobe that grows optimally at 100 degrees C by a fermentative-type metabolism in which H2 and CO2 are the only detectable products. A ferredoxin, which functions as the electron donor to the hydrogenase of this organism was purified under anaerobic reducing conditions. It had a molecular weight of approximately 12,000 and contained 8 iron atoms and 8 cysteine residues/mol but lacked histidine or arginine residues. Reduction and oxidation of the ferredoxin each required 2 electrons/mol, which is consistent with the presence of two [4Fe-4S] clusters. The reduced protein gave rise to a broad rhombic electronic paramagnetic resonance spectrum, with gz = 2.10, gy = 1.86, gx = 1.80, and a midpoint potential of -345 mV (at pH 8). However, this spectrum represented a minor species, since it quantitated to only approximately 0.3 spins/mol. P. furiosus ferredoxin is therefore distinct from other ferredoxins in that the bulk of its iron is not present as iron-sulfur clusters with an S = 1/2 ground state. The apoferredoxin was reconstituted with iron and sulfide to give a protein that was indistinguishable from the native ferredoxin by its iron content and electron paramagnetic resonance properties, which showed that the novel iron-sulfur clusters were not artifacts of purification. The reduced ferredoxin also functioned as an electron donor for H2 evolution catalyzed by the hydrogenase of the mesophilic eubacterium Clostridium pasteurianum. P. furiosus ferredoxin was resistant to denaturation by sodium dodecyl sulfate (20%, wt/vol) and was remarkably thermostable. Its UV-visible absorption spectrum and electron carrier activity to P. furiosus hydrogenase were unaffected by a 12-h incubation of 95 degrees C.     

Involvement of protein kinase C in platelet-activating factor-stimulated diacylglycerol accumulation in murine peritoneal macrophages

Incubation of murine peritoneal macrophages with platelet-activating factor (PAF; 1-O-alkyl(C16 + C18)-2-acetyl-sn-glycerol-3-phosphorylcholine) results in the rapid accumulation of [3H]inositol phosphates and sn-1,2-diacylglycerol (DAG) and mobilization of intracellular calcium (Prpic, V., Uhing, R. J., Weiel, J. E., Jakoi, L., Gawdi, G., Herman, B., and Adams, D. O. (1988) J. Cell Biol. 107, 363-372). We have further investigated the relationship of phosphoinositide metabolism to accumulation of DAG and the possible involvement of protein kinase C in the accumulation of DAG in response to PAF. DAG accumulation proceeds at a slower rate than the accumulation of either [3H] inositol 1,4,5-trisphosphate or total [3H]inositol phosphates. Accumulation of DAG from additional precursors is suggested from both an estimation of the mass of total inositol phosphates produced and the accumulation of [3H]choline in response in PAF. Down-regulation of protein kinase C by prolonged pretreatment with phorbol ester or inhibition of the enzyme with sphingosine inhibited the PAF-generated accumulation of DAG at 10 min by approximately 80%. Under the same conditions, no inhibition of PAF-stimulated generation of [3H]inositol 1,4,5-trisphosphate was observed. Similar inhibition was observed when 10 microM ionomycin or 0.1 microM phorbol 12-myristate 13-acetate were used to stimulate accumulation of DAG. The results suggest that PAF stimulates the accumulation of DAG from source other than phosphatidylinositol metabolism in peritoneal macrophages and that this occurs subsequent to the activation of protein kinase C.     

Immunohistochemical localization of vimentin in the gerbil inner ear

Cells containing immunoreactive vimentin-type intermediate filaments (IF) were identified in paraffin sections and whole-mount preparations of the gerbil inner ear. Most connective tissue cells showed positive immunostaining, although one unusual class of stromal cell lacked vimentin. Several different types of epithelial cells contained high levels of vimentin. In the cochlea, Deiters' cells, inner phalangeal cells, Boettcher's cells, some outer sulcus cells, and the intermediate cells of the stria vascularis showed strong immunoreactivity. Strial basal cells exhibited weaker and less consistent staining. Neither inner nor outer hair cells were stained. In the vestibular system, hair cells with a morphology and location more characteristic of type I than of type II cells showed strong immunostaining for vimentin. Supporting cells in vestibular neurosensory epithelium stained with less intensity. These results were surprising because epithelial cells in vivo only rarely express vimentin-type IF. Although the functional significance of vimentin remains to be established, its presence in some but not other highly specialized cell types provides an excellent marker for investigating the lineage and morphogenesis of the complex inner ear tissues.     

Photosynthesis and Chlorophyll Fluorescence Characteristics in Relationship to Changes in Pigment and Element Composition of Leaves of Platanus occidentalis L. during Autumnal Leaf Senescence

The loss of chlorophyll and total leaf nitrogen during autumnal senescence of leaves from the deciduous tree Platanus occidentalis L. was accompanied by a marked decline in the photosynthetic capacity of O₂ evolution on a leaf area basis. When expressed on a chlorophyll basis, however, the capacity for light-and CO₂-saturated O₂ evolution did not decline, but rather increased as leaf chlorophyll content decreased. The photon yield of O₂ evolution in white light (400-700 nanometers) declined markedly with decreases in leaf chlorophyll content below 150 milligrams of chlorophyll per square meter on both an incident and an absorbed basis, due largely to the absorption of light by nonphotosynthetic pigments which were not degraded as rapidly as the chlorophylls. Photon yields measured in, and corrected for the absorptance of, red light (630-700 nanometers) exhibited little change with the loss of chlorophyll. Furthermore, PSII photochemical efficiency, as determined from chlorophyll fluorescence, remained high, and the chlorophyll a/b ratio exhibited no decline except in leaves with extremely low chlorophyll contents. These data indicate that the efficiency for photochemical energy conversion of the remaining functional components was maintained at a high level during the natural course of autumnal senescence, and are consistent with previous studies which have characterized leaf senescence as being a controlled process. The loss of chlorophyll during senescence was also accompanied by a decline in fluorescence emanating from PSI, whereas there was little change in PSII fluorescence (measured at 77 Kelvin), presumably due to decreased reabsorption of PSII fluorescence by chlorophyll. Nitrogen was the only element examined to exhibit a decline with senescence on a dry weight basis. However, on a leaf area basis, all elements (C, Ca, K, Mg, N, P, S) declined in senescent leaves, although the contents of sulfur and calcium, which are not easily retranslocated, decreased to the smallest extent.     

Colorimetric enumeration of Escherichia coli based on beta-glucuronidase activity

A medium containing a chromogenic substrate was developed for the enumeration of Escherichia coli on the basis of beta-glucuronidase activity. In this medium there was an inverse linear relationship between the log initial E. coli concentration and the time taken for the color to reach a threshold optical density of 0.05. This relationship applied even when the E. coli population contained 5% beta-glucuronidase-negative cells. Incubation at 44 degrees C reduced the time taken for color development and allowed the procedure to be used in the presence of a competitive microflora that outnumbered the E. coli population by a factor of 10(4). Sodium lauryl sulfate as an additional selective agent gave no significant improvement. In the analysis of environmental water samples, the technique gave a good correlation with a standard cultural method. The procedure shows promise as a simple method for testing the compliance of environmental samples with microbiological criteria for E. coli.     

The ontogeny of immunoreactive beta-endorphin and beta-lipotropin in the rat testis

We have investigated the ontogeny of immunoreactive beta-endorphin (i-beta E) in the testes of rats from 5 to 150 days of age. i-beta E was measured by RIA in acid extracts of decapsulated testes and characterized by gel filtration chromatography. Significant age-related differences in both the levels and type of i-beta E were observed. Total levels of i-beta E in the testes were very low and barely detectable from 5-20 days of age, but rose sharply in parallel with testes weights from 20-60 days of age; thereafter, no significant changes in i-beta E were found through 150 days of age. Concentrations of i-beta E, expressed in pmol/g testis, fell precipitously between days 5 and 10 and remained relatively constant from 10-150 days. Most of the i-beta E at 5 and 15 days chromatographed like authentic beta-endorphin. However, with the onset of puberty (30-35 days) and during sexual maturation, much of the total i-beta E chromatographed like its' precursor beta-lipotropin (beta LPH). Hypophysectomy decreased the weight and total i-beta E levels of the testes to the same extent without altering the concentrations of i-beta E or the chromatographic pattern of i-beta E. These results indicate that beta E-like and beta LPH-like peptides are present in the rat testis and that age-related changes in both the levels and type of i-beta E correlate with various structural and functional aspects of testicular development.     

Characterization of an extremely thermostable glutamate dehydrogenase: a key enzyme in the primary metabolism of the hyperthermophilic archaebacterium, Pyrococcus furiosus.

Glutamate dehydrogenase (L-glutamate:NAD(P)+ oxidoreductase, deaminating, EC 1.4.1.3) from the hyperthermophilic Archeon Pyrococcus furiosus was purified to homogeneity by chromatography on anion-exchange, molecular-exclusion and hydrophobic-interaction media. The purified native enzyme had an M(r) of 270,000 +/- 15,000 and was shown to be a hexamer with identical subunits of M(r) 46,000. The enzyme was exceptionally thermostable, having a half-life of 3.5 to more than 10 h at 100 degrees C, depending on the concentration of enzyme. The Km of the enzyme for ammonia was high (9.5 mM), indicating that the enzyme is probably active in the deaminating, catabolic direction. The coenzyme utilization of the enzyme resembled the equivalent enzymes from eukaryotes rather than eubacteria, since both NADH and NADPH were recognized with high affinity. The enzyme displayed a preference for NADP+ over NAD+ that was more pronounced at low assay temperatures (50-70 degrees C) compared with the optimal temperature for enzyme activity, 95 degrees C.     

The carotenoid zeaxanthin and ‘high-energy-state quenching’ of chlorophyll fluorescence

The possibility that zeaxanthin mediates the dissipation of an excess of excitation energy in the antenna chlorophyll of the photochemical apparatus has been tested through the use of an inhibitor of violaxanthin de-epoxidation, dithiothreitol (DTT), as well as through the comparison of two closely related organisms (green and blue-green algal lichens), one of which (blue-green algal lichen) naturally lacks the xanthophyll cycle. In spinach leaves, DTT inhibited a major component of the rapidly relaxing high-energy-state quenching' of chlorophyll fluorescence, which was associated with a quenching of the level of initial fluorescence (F₀) and exhibited a close correlation with the zeaxanthin content of leaves when fluorescence quenching was expressed as the rate constant for radiationless energy dissipation in the antenna chlorophyll. Green algal lichens, which possess the xanthophyll cycle, exhibited the same type of fluorescence quenching as that observed in leaves. Two groups of blue-green algal lichens were used for a comparison with these green algal lichens. A group of zeaxanthin-free blue-green algal lichens did not exhibit the type of chlorophyll fluorescence quenching indicative of energy dissipation in the pigment bed. In contrast, a group of blue-green algal lichens which had formed zeaxanthin slowly through reactions other than the xanthophyll cycle, did show a very similar response to that of leaves and green algal lichens. Fluorescence quenching indicative of radiationless energy dissipation in the antenna chlorophyll was the predominant component of high-energy-state quenching in spinach leaves under conditions allowing for high rates of steady-state photosynthesis. A second, but distinctly different type of high-energy-state quenching of chlorophyll fluorescence, which was not inhibited by DTT (i.e., it was zeaxanthin independent) and which is possibly associated with the photosystem II reaction center, occurred in addition to that associated with zeaxanthin in leaves under a range of conditions which were less favorable for linear photosynthetic electron flow. In intact chloroplasts isolated from (zeaxanthin-free) spinach leaves a combination of these two types of rapidly reversible fluorescence quenching occurred under all conditions examined.Abbreviations DTT dithiothreitol - F₀ (or F₀) yield of instantaneous fluorescence at open PS II reaction centers in the dark (or during actinic illumination) - F_M (or F_M) yield of maximum fluorescence induced by a saturation pulse of light in the dark (or during actinic illumination) - F_V (or F_V) yield of variable fluorescence induced by a saturating pulse of light in the dark (or during actinic illumination) - k _D rate constant for radiationless energy dissipation in the antenna chlorophyll - SV Stern-Volmer equation - PFD photon flux density - PS I photosystem I - PS II photosystem II - Q_A acceptor of photosystem II - q_N coefficient of nonphotochemical chlorophyll fluorescence quenching - q_P coefficient of photochemical chlorophyll fluorescence quenching     

Ventilatory control during exercise in calves with artificial hearts

To determine the role of cardiac reflexes in mediating exercise hyperpnea, we investigated ventilatory responses to treadmill exercise in seven calves with artificial hearts and seven controls. In both groups, the ventilatory responses were adequate for the metabolic demands of the exercise; this resulted in regulation of arterial PCO2 and pH despite the absence of cardiac output increase in the implanted group. In this group, there was a small but significant reduction of arterial PO2 by 4 +/- 3 Torr and a rise of blood lactate by 1.1 +/- 1 mmol/l. When cardiac output was experimentally increased in the implanted calves to a level commensurate with that spontaneously occurring in the control calves, ventilation was not affected. However, experimental reductions of cardiac output led to an immediate augmentation of exercise hyperpnea by 4.56 +/- 4.3 l/min and a further significant lactate increase of 1.2 +/- 1.22 mmol/l that was associated with a significant decrease in the exercise O2 consumption (0.32 +/- 0.13 l/min). These observations indicate that neither cardiac nor hemodynamic effects of increased cardiac output constitute an obligatory cause of exercise hyperpnea in the calf.     

Molecular and phylogenetic characterization of pyruvate and 2-ketoisovalerate ferredoxin oxidoreductases from Pyrococcus furiosus and pyruvate ferredoxin oxidoreductase from Thermotoga maritima.

Previous studies have shown that the hyperthermophilic archaeon Pyrococcus furiosus contains four distinct cytoplasmic 2-ketoacid oxidoreductases (ORs) which differ in their substrate specificities, while the hyperthermophilic bacterium Thermotoga maritima contains only one, pyruvate ferredoxin oxidoreductase (POR). These enzymes catalyze the synthesis of the acyl (or aryl) coenzyme A derivative in a thiamine PPi-dependent oxidative decarboxylation reaction with reduction of ferredoxin. We report here on the molecular analysis of the POR (por) and 2-ketoisovalerate ferredoxin oxidoreductase (vor) genes from P. furiosus and of the POR gene from T. maritima, all of which comprise four different subunits. The operon organization for P. furiosus POR and VOR was porG-vorDAB-porDAB, wherein the gamma subunit is shared by the two enzymes. The operon organization for T. maritima POR was porGDAB. The three enzymes were 46 to 53% identical at the amino acid level. Their delta subunits each contained two ferredoxin-type [4Fe-4S] cluster binding motifs (CXXCXXCXXXCP), while their beta subunits each contained four conserved cysteines in addition to a thiamine PPi-binding domain. Amino-terminal sequence comparisons show that POR, VOR, indolepyruvate OR, and 2-ketoglutarate OR of P. furiosus all belong to a phylogenetically homologous OR family. Moreover, the single-subunit pyruvate ORs from mesophilic and moderately thermophilic bacteria and from an amitochondriate eucaryote each contain four domains which are phylogenetically homologous to the four subunits of the hyperthermophilic ORs (27% sequence identity). Three of these subunits are also homologous to the dimeric POR from a mesophilic archaeon, Halobacterium halobium (21% identity). A model is proposed to account for the observed phenotypes based on genomic rearrangements of four ancestral OR subunits.