Sequence analysis reveals new membrane anchor of reaction centre-bound cytochromes possibly related to PufX

Most of the bacterial photosynthetic reaction centres known to date contain a cytochrome subunit with four covalently bound haem groups. In the case of Blastochloris viridis, this reaction centre subunit is anchored in the membrane by a lipid molecule covalently attached to the cysteine which forms the N-terminus of the mature protein after processing by a signal peptidase. We show that posttranslational N-terminal cleavage of the cytochrome subunit does not occur in the aerobic photosynthetic bacterium Roseobacter denitrificans. From sequence analysis of the resulting elongated N-terminus it follows that a transmembrane helix is anchoring the reaction centre-bound cytochrome in the membrane. Comparative sequence analysis strongly suggests that all cytochrome subunits lacking the lipid coupling cysteine share this structural feature. Comparison of the N-terminal segment of the cytochrome subunit of Roseobacter denitrificans with the sequences of the PufX proteins from Rhodobacter sphaeroides and Rhodobacter capsulatus suggests a phylogenetic relation.     

Characterization of green seed,an enhancer of abi3-1 in Arabidopsis that affects seed longevity

Seeds are usually stored in physiological conditions in which they gradually lose their viability and vigor depending on storage conditions, storage time, and genotype. Very little is known about the underlying genetics of seed storability and seed deterioration. We analyzed a mutant in Arabidopsis disturbed in seed storability. This mutant was isolated as a grs (green-seeded) mutant in an abi3-1 (abscisic acid 3) mutant background. Genetic and physiological characterization showed that the monogenic grs mutant was not visibly green seeded and mapped on chromosome 4. This enhancer mutation did not affect the ABA sensitivity of seed germination or seed dormancy but was found to affect seed storability and seedling vigor. Seed storability was assessed in a controlled deterioration test, in which the germination capacity of the mutant decreased with the duration of the treatment. The decrease in viability and vigor was confirmed by storing the seeds in two relative humidities (RHs) for a prolonged period. At 60% RH, the mutant lost germinability, but storage at 32% RH showed no decrease of germination although seed vigor decreased. The decrease in viability and vigor could be related to an increase in conductivity, suggesting membrane deterioration. This was not affected by light conditions during imbibition, expected to influence the generation of active oxygen species. During seed maturation, ABI3 regulates several processes: acquiring dormancy and long-term storability and loss of chlorophyll. Our results indicate that GRS is a common regulator in the latter two but not of dormancy/germination.     

Reversal of cyanide inhibition of cytochrome c oxidase by the auxiliary substrate nitric oxide: an endogenous antidote to cyanide poisoning?

Nitric oxide (NO) is shown to overcome the cyanide inhibition of cytochrome c oxidase in the presence of excess ferrocytochrome c and oxygen. Addition of NO to the partially reduced cyanide-inhibited form of the bovine enzyme is shown by electron paramagnetic resonance spectroscopy to result in substitution of cyanide at ferriheme a3 by NO with reduction of the heme. The resulting nitrosylferroheme a3 is a 5-coordinate structure, the proximal bond to histidine having been broken. NO does not simply act as a reversibly bound competitive inhibitor but is an auxiliary substrate consumed in a catalytic cycle along with ferrocytochrome c and oxygen. The implications of this observation with regard to estimates of steady-state NO levels in vivo is discussed. Given the multiple sources of NO available to mitochondria, the present results appear to explain in part some of the curious biomedical observations reported by other laboratories; for example, the kidneys of cyanide poisoning victims surprisingly exhibit no significant irreversible damage, and lethal doses of potassium cyanide are able to inhibit cytochrome c oxidase activity by only approximately 50% in brain mitochondria.     

Structure and surface energy of the surfactant layer on the alveolar surface: inaccuracies and their correction

The article of Kashchiev and Exerowa (Eur Biophys J 30:34-41) is shown to incorporate a number of inaccuracies that fit the categories "historical", "anatomical", and "biophysical". These inaccuracies are corrected by reference to published research reports from 1978 to 1998. The monolayer-bilayer model proposed by Kashchiev and Exerowa may be thermodynamically correct in vitro, but has not been related to the structure of the alveolar surface in vivo, which is that of a foam ("the alveolar surface network").     

Molecular and biochemical characterization of a distinct type of fructose-1,6-bisphosphatase from Pyrococcus furiosus

The Pyrococcus furiosus fbpA gene was cloned and expressed in Escherichia coli, and the fructose-1,6-bisphosphatase produced was subsequently purified and characterized. The dimeric enzyme showed a preference for fructose-1,6-bisphosphate, with a K(m) of 0.32 mM and a V(max) of 12.2 U/mg. The P. furiosus fructose-1,6-bisphosphatase was strongly inhibited by Li(+) (50% inhibitory concentration, 1 mM). Based on the presence of conserved sequence motifs and the substrate specificity of the P. furiosus fructose-1,6-bisphosphatase, we propose that this enzyme belongs to a new family, class IV fructose-1,6-bisphosphatase.     

Functional expression and mutational analysis of flavonol synthase from Citrus unshiu.

Flavonols are produced by the desaturation of flavanols catalyzed by flavonol synthase. The enzyme belongs to the class of intermolecular dioxygenases which depend on molecular oxygen and FeII/2-oxoglutarate for activity, and have been in focus of structural studies recently. Flavonol synthase cDNAs were cloned from six plant species, but none of the enzymes had been studied in detail. Therefore, a cDNA from Citrus unshiu (Satsuma mandarin) designated as flavonol synthase was expressed in Escherichia coli, and the purified recombinant enzyme was subjected to kinetic and mutational chacterizations. The integrity of the recombinant synthase was revealed by a molecular ion from MALDI-TOF mass spectrometry at m/z 37888 +/- 40 (as compared to 37899 Da calculated for the translated polypeptide), and by partial N-terminal sequencing. Maximal flavonol synthase activity was observed in the range of pH 5-6 with dihydroquercetin as substrate and a temperature optimum at about 37 degrees C. Km values of 272, 11 and 36 micro m were determined for dihydroquercetin, FeII and 2-oxoglutarate, respectively, with a sixfold higher affinity to dihydrokaempferol (Km 45 micro m). Flavonol synthase polypeptides share an overall sequence similarity of 85% (47% identity), whereas only 30-60% similarity were apparent with other dioxygenases. Like the other dioxygenases of this class, Citrus flavonol synthase cDNA encodes eight strictly conserved amino-acid residues which include two histidines (His221, His277) and one acidic amino acid (Asp223) residue for FeII-coordination, an arginine (Arg287) proposed to bind 2-oxoglutarate, and four amino acids (Gly68, His75, Gly261, Pro207) with no obvious functionality. Replacements of Gly68 and Gly261 by alanine reduced the catalytic activity by 95%, while the exchange of these Gly residues for proline completely abolished the enzyme activity. Alternatively, the substitution of Pro207 by glycine hardly affected the activity. The data suggest that Gly68 and Gly261, at least, are required for proper folding of the flavonol synthase polypeptide.     

Trehalose turnover and the coexistence of trehalose and active trehalase in cockroach haemolymph

In the cockroaches Periplaneta americana, Periplaneta australasiae, Leucophaea maderae, and Nauphoeta cinerea, undiluted haemolymph, undiluted haemolymph to which 10% solid trehalose was added, and haemolymph diluted 100 or more times with 1% trehalose solution showed approximately equal trehalase activities (3 to 8 mg/ml per hr). No evidence for the presence of a trehalase inhibitor was found.Freshly drawn haemolymph of Periplaneta americana contained 14 to 16 mg trehalose/ml, which on standing was hydrolyzed to glucose at a rate of 4 to 8 mg/ml per hr. In this cockroach, the rate of haemolymph trehalose turnover was only 1.3 mg/ml per hr. This means that in vitro trehalose is hydrolyzed by undiluted haemolymph at several times the rate at which it is replaced in the haemolymph of the intact insect. The mechanism through which trehalose and trehalase can coexist in the haemolymph of the intact cockroach remains therefore unexplained.     

Revisiting junk DNA

Summary The distribution of functions within genomes of higher organisms relative to processes that lead to the spread of mutations in populations is examined in its general outlines. A number of points are enumerated that collectively put in question the concept of junk DNA: the plausible compatibility of DNA function with rapid substitution rates; the likelihood of superimposed functions along much of eukaryotic DNA; the potential for a merely conditional functionality in sequence repeats; the apparent adoption of macromolecular waste as a strategy for maintaining a function without selective grooming of individual sequence repeats that carry out the function; the likely requirement that any DNA sequence must be polite vis-à-vis (compatible with) functional sequences in its genomic environment; the existence in germ-cell lineages of selective constraints that are not apparent in populations of individuals; and the fact that DNA tectonics-the appearance and disappearance of genomic DNA-are not incompatible with function. It is pointed out that the inverse correlation between functional constraints and rates of substitution cannot be claimed to be a pillar of the neutral theory, because it is also predicted from a selectionist view-point. The dispensability of functional structures is brought into relation with the concept of reproductive sufficiency-the survivability of genotypes in the absence of fitter alleles.     

Lipolytic and adenyl-cyclase-stimulating activity of glucagon1–6: comparison with glucagon derivatives chemically modified in the 7–29 sequence

Glucagon_1–6 has a maximum lipolytic activity (L_max) in the rat adipocyte which is 66% of that of glucagon. The N-guanidyl derivative, modified at Lys₁₂ , has about the same L_max as glucagon_1–6. Modifying the carboxyl groups of glucagon with glycinamide or removing the COON-terminal residues with cyanogen bromide reduces L_max to less than 25% of the level of glucagon. The potency of each of these analogs (A₅₀) in M is as follows: glucagon 6×10^–3; glucagon^1–6 2 ×10^–2; N-guanidyl glucagon 9×10^–3; glycinamide glucagon 10^–2; cyanogen bromide peptide of glucagon 2 ×10^–1. The ability of all of the glucagon analogs to stimulate adenyl cyclase was somewhat less than their tipolytic activities with the exception of the glycin-amide derivative and the cyanogen bromide peptide, which were slightly more active in stimulating adenyl cyclase than in lipolysis. Glucagon_1–6 is much more potent in stimulating adipocyte than liver adenyl cyclase.     

Human Exposure to Wild Animals in the Sankuru Province of the Democratic Republic of the Congo

Due to the high level of biological diversity in the Congo Basin and human population dependence on bushmeat, the DRC represents an ideal location for expanding knowledge on wild animal exposures and thus the potential for transmission of zoonotic pathogens. However, limited information exists on patterns and extent of contact with wildlife in such communities. Using a cross-sectional study, 14 villages in the Sankuru Province of the DRC were surveyed between August and September 2007. Villagers ≥ 1 year of age and at home of the time of the survey were eligible and enrolled to describe and assess factors associated with animal exposures (both activity and type of animal). Among respondents, 91% reported exposure to rodents, 89% to duikers, 78% to non-human primates (NHPs), and 32% reported contact with bats in the month prior to the survey. The most frequently reported activities included eating (95%), cooking (70%), and butchering or skinning of animals (55%). The activities and animals to which subjects had contact varied by sex and age. Moreover, we observed a high correlation of the same activities across animal types. In this and other populations that rely on bushmeat, there is a high frequency of exposure to multiple animal species through various modalities. In the event of future zoonotic disease outbreaks, effective public health interventions and campaigns that mitigate the risk of animal contact during outbreaks need to be broad to include various modes of contact and should be directed to both men and women across all age groups. As available information is limited, further studies are necessary to better understand the complex relationships and exposures individuals have with animals.