Redox intermediates of Desulfovibrio gigas [NiFe] hydrogenase generated under hydrogen. M?ssbauer and EPR characterization of the metal centers

The hydrogenase (EC 1.2.2.1) of Desulfovibrio gigas is a complex enzyme containing one nickel center, one [3Fe-4S] and two [4Fe-4S] clusters. Redox intermediates of this enzyme were generated under hydrogen (the natural substrate) using a redox-titration technique and were studied by EPR and M?ssbauer spectroscopy. In the oxidized states, the two [4Fe-4S]2+ clusters exhibit a broad quadrupole doublet with parameters (apparent delta EQ = 1.10 mm/s and delta = 0.35 mm/s) typical for this type of cluster. Upon reduction, the two [4Fe-4S]1+ clusters are spectroscopically distinguishable, allowing the determination of their midpoint redox potentials. The cluster with higher midpoint potential (-290 +/- 20 mV) was labeled Fe-S center I and the other with lower potential (-340 +/- 20 mV), Fe-S center II. Both reduced clusters show atypical magnetic hyperfine coupling constants, suggesting structural differences from the clusters of bacterial ferredoxins. Also, an unusually broad EPR signal, labeled Fe-S signal B', extending from approximately 150 to approximately 450 mT was observed concomitantly with the reduction of the [4Fe-4S] clusters. The following two EPR signals observed at the weak-field region were tentatively attributed to the reduced [3Fe-4S] cluster: (i) a signal with crossover point at g approximately 12, labeled the g = 12 signal, and (ii) a broad signal at the very weak-field region (approximately 3 mT), labeled the Fe-S signal B. The midpoint redox potential associated with the appearance of the g = 12 signal was determined to be -70 +/- 10 mV. At potentials below -250 mV, the g = 12 signal began to decrease in intensity, and simultaneously, the Fe-S signal B appeared. The transformation of the g = 12 signal into the Fe-S signal B was found to parallel the reduction of the two [4Fe-4S] clusters indicating that the [3Fe-4S]o cluster is sensitive to the redox state of the [4Fe-4S] clusters. Detailed redox profiles for the previously reported Ni-signal C and the g = 2.21 signal were obtained in this study, and evidence was found to indicate that these two signals represent two different oxidation states of the enzyme. Finally, the mechanistic implications of our results are discussed.     

CAP 37, a 37 kD human neutrophil granule cationic protein shares homology with inflammatory proteinases

We have previously shown that a major granule-associated cationic protein CAP 37 (Mr = 37 kD) derived from human PMN is a monocyte-specific chemoattractant. The N-terminal amino acid sequence of this novel chemotactic protein shares significant homology with a number of inflammatory molecules with protease activity including elastase and cathepsin G. However, a critical substitution of a serine for a histidine at position 41, results in its lack of serine protease activity.     

Iron deficiency in an area of Bolivia and high prevalence of intestinal helminthiasis

Between November and December 1987, 201 subjects from three communities of the Cordillera Province, Santa Cruz Department, South-East Bolivia (Camiri, Boyuibe, and Gutierrez) were examined for serum iron levels. Sixty-three (31.3%) were iron deficient. There were no significant differences between males and females (37.2% and 26.9%, respectively) and among the three localities studied. Iron deficiency was highest in the 3- to 9-year-old age group and lowest in the 30- to 39-year-old group. Considering the prevalence in the same localities of infections by hookworms, Trichuris trichiura, Strongyloides stercoralis, and Trichostrongylus, it is suggested that hookworms and possibly other intestinal parasites may be an important cause of the observed iron deficiency.     

The 56 kDa protein of human genital skin fibroblasts is identical to that radiolabelled by [3H]dihydrotestosterone 17 beta-bromoacetate

Analysis of soluble proteins from human genital skin fibroblasts by two-dimensional polyacrylamide gel electrophoresis reveals an abundant protein doublet of mol. wt 56,000 with isoelectric points (pI) of 6.7 and 6.5. This protein is absent in non-genital skin fibroblasts as well as in genital skin fibroblasts of most patients with complete forms of androgen insensitivity. The protein specifically binds androgen. A protein of similar estimated molecular weight (58,000) from human genital skin fibroblasts has recently been found to be covalently radiolabelled by the affinity ligand dihydrotestosterone 17 beta-bromoacetate (DHT-BA). In the present study these proteins have been found to be indistinguishable on one- and two-dimensional gel electrophoresis. Antibodies raised against the 56 kDa pI 6.7/6.5 protein also recognized the protein covalently radiolabelled by DHT-BA. A third protein of estimated mol. wt 59,000 has been found to be associated with several steroid hormone receptor complexes but has no known ligand binding activity. This protein was found to be clearly separable from the 56/58 kDa protein on two-dimensional gel electrophoresis as it has a more acidic pI of approximately 5.4. Furthermore, antibodies against the 59 kDa protein do not recognize the 56 kDa species, and vice versa.     

An antibody against a CFTR-derived synthetic peptide, incorporated into living submandibular cells, inhibits beta-adrenergic stimulation of mucin secretion.

An antibody raised against a peptide in the first nucleotide-binding domain (NBD) of CFTR [1], incorporated into intact rat submandibular acini by hypotonic swelling, inhibited beta-adrenergic stimulated mucin secretion, without affecting cyclic AMP rise. The data are the first to show that a CFTR-antibody-containing cell results in defective stimulation of mucin secretion, as is seen in CF cells, and that this can be reversed by an excessive increase in cyclic AMP.     

GROWTH PATTERN,REPRODUCTION, AND SELF-THINNING IN SEAWEEDS1

In unispecific plant stands, the logarithm of mean individual weight (w) depends on the logarithm of density (d) by the -3/4 power law (a slope of -1.5 and an intercept ranging from 2.3 to 5.0). The analysis of the w and d relationships in whole cohorts of two seaweed species from the Strait of Gibraltar shows deviations from the canonical equation. The kelp Phyllariopsis purpurascens (C. Agardh) Henry et South (Phaeophyta) growing at a 30-m depth has the lowest intercept value (0.6) recorded for any plant species and a slope not significantly different from -1.5. The slope value is in accordance with those found in species whose growth is not stopped by reproduction. Irradiance under a single layer of blades was lower than the photosynthetic light compensation point, and this could be due to overdispersion of the shoots and, in consequence, to the low intercept value of the self-thinning equation. The w to d relationships in Asparagopsis armata Harvey (Rhodophyta) show two different components: no dependence between these two variables (slope not significantly different from 0) at densities < 500 shoots·m^?2, and a slope more negative (-2.1) than proposed by the -3/2 power law at densities > 500 shoots·m^?2. The pattern at high densities could be due to intraspecific competition for light, whereas the slope ～0 at low densities could be related to inhibition of growth by reproduction (cystocarp and carpospore production). Therefore, rather than being considered exceptional, we suggest that a gradient of variability could be expected in the dependence of w on d when specific growth patterns and reproduction are considered.     

Small-scale association measures in epibenthic communities as a clue for allelochemical interactions

The small-scale associations in a rocky subtidal community in the northwestern Mediterranean were studied by a development of the continuous line transect method. This method allowed the overall measurement of non-randomness in interspecific contacts and the assignment of an association index to each species-pair, whose, significance was tested by Monte Carlo procedures. At the same time, the continuous recording allowed the study of the weakening of the interactions with increasing distances. Our purpose was to uncover evidence for allelochemical mechanisms of space occupation and maintenance. A strong non-randomness was found in the interspecific associations. This was mostly due to the interactions of the poecilosclerid sponge Crambe crambe (Schmidt) with its neighbours, especially its negative associations with other sponge species. The strength of the relationships fell drastically over the first few centimeters from the contact borders of the different species. The results pointed strongly to an allelochemical mechanism. The extracts of this sponge featured high bioactivity in laboratory assays, and field experiments demonstrated that the sponge can inhibit the growth of species in the community studied. Standard sampling techniques would have overlooked the spatial structure present in the data. The study emphasizes the need for both contact data and distance data in order to identify the underlying processes reliably. The line transect method provides both types of information easily and allows testing of models and identification of organisms likely to use chemical defenses in space competition. Its use as a preliminary step in studies of chemical ecology might help to detect presumptive allelochemical processes prior to experimental work on the potentially active species.     

A PCR-based test suitable for screening for fragile X syndrome among mentally retarded males

Ever since the identification of the genetic cause of fragile X syndrome as the expansion of an unstable trinucleotide sequence, several diagnostic strategies have evolved from molecular studies. However, we still lack a simple test suitable for population screening. We have therefore developed a nonisotopic polymerase chain reaction (PCR)-based technique for the identification of fragile X full mutations among men, with easy visualization of the PCR products on silver-stained polyacrylamide gels. The technique consists of PCR amplification with primers that flank the trinucleotide repeats, with a product of 557 bp for the (CGG)₂₉ allele. Conditions were established such that full mutations failed to amplify and were thus identified with 98% sensitivity compared with Southern blot analysis. To produce an indispensable internal control we added to the reaction a third primer, internal to this fragment, allowing the multiplex amplification of a monomorphic band corresponding to a CG-rich stretch 147 bp upstream of the polymorphic region. In trials involving 41 patients and 74 controls, the PCR-based test here described showed specificity of more than 98.6%, accuracy of 99% and a sensitivity of 98%. Thus, although not suitable for medical diagnosis, it constitutes a useful tool for screening for the fragile X syndrome in populations of mentally retarded males. Received: 31 May 1995 / Revised: 4 October 1995     

Colonization and deterioration processes in Roman mortars by cyanobacteria,algae and lichens

The mortars covering some walls of the Roman city of Baelo Claudia (Cadiz, Spain) support an abundant colonization of cyanobacteria, algae and lichens. The distribution of these organisms is closely related to microclimatic parameters. Furthermore, the development, specific composition and biomass of algal cryptoendolithic communities are related to the wall orientation. The effect of these communities on mortar deterioration is discussed.     

Variable influence of ferric and cupric ions on Saccharomyces cerevisiae strains used in asymmetric organic synthesis

Summary Fe (III) and Cu (II) each at 50 M in four commercial strains of Saccharomyces cerevisiae induced an increase of NAD(P)⁺ reduction in one strain (Turkish), but two others (Chilean and Brazilian), the presence of Fe(III) and/or Cu(II) diminished NAD(P)⁺ reduction presumably due to free radicais formation inside these living cells. Suprisingly, in the American strain, Fe(III) induced a decrease and Cu (II) an increase of NAD(P)⁺ reduction.