Organization of the genes necessary for hydrogenase expression in Rhodobacter capsulatus. Sequence analysis and identification of two hyp regulatory mutants

A 25kbp DNA fragment from the chromosome of Rhodobacter capsulatus B10 carrying hydrogenase (hup) determinants was completely sequenced. Coding regions corresponding to 20 open reading frames were identified. The R. capsulatus hydrogenase-specific gene (hup and hyp) products bear significant structural identity to hydrogenase gene products from Escherichia coli (13), from Rhizobium liguminosarum (16), from Azotobacter vinelandii (10) and from Alcaligenes eutrophus (11). The sequential arrangement of the R. capsulatus genes is: hupR2-hupU-hypF -hupS-hupL-hupM-hupD -hupF -hupG -hupH -huoJ -hupK -hypA-hypB-hupR1-hypC -hypD -hypE -ORF19 -ORF20 , all contiguous and transcribed from the same DNA strand. The last two potential genes do not encode products that are related to identified hydrogenase-specific gene products in other species. The sequence of the 12 R. capsulatus genes underlined above is presented. The mutation site in two of the Hup^? mutants used in this study, RS13 and RCC12, was identified in the hypF gene (deletion of one G) and in the hypD qene (deletion of 54 bp), respectively. The hypF gene product shares 45% identity with the product of hydA from E. coli and the product of hypF from R. leguminosarum. Those products present at their N-terminus a Cys arrangement typical of zinc-finger proteins. The G deletion in the C-terminal region of hypF in the RS13 mutant     

Selection of synthetic proteins to modulate the human frataxin function

Frataxin is a kinetic activator of the mitochondrial supercomplex for iron-sulfur cluster assembly. Low frataxin expression or a decrease in its functionality results in Friedreich's Ataxia (FRDA). With the aim of creating new molecular tools to study this metabolic pathway, and ultimately, to explore new therapeutic strategies, we have investigated the possibility of obtaining small proteins exhibiting a high affinity for frataxin. In this study, we applied the ribosome display approach, using human frataxin as the target. We focused on Affi_224, one of the proteins that we were able to select after five rounds of selection. We have studied the interaction between both proteins and discussed some applications of this specific molecular tutor, concerning the modulation of the supercomplex activity. Affi_224 and frataxin showed a K_D value in the nanomolar range, as judged by surface plasmon resonance analysis. Most likely, it binds to the frataxin acidic ridge, as suggested by the analysis of chemical shift perturbations (nuclear magnetic resonance) and computational simulations. Affi_224 was able to increase Cys NFS1 desulfurase activation exerted by the FRDA frataxin variant G130V. Importantly, Affi_224 interacts with frataxin in a human cellular model. Our results suggest quaternary addition may be a new tool to modulate frataxin function in vivo. Nevertheless, more functional experiments under physiological conditions should be carried out to evaluate Affi_224 effectiveness in FRDA cell models.     

Same species,different population dynamics: Spatio-temporal differences of Undaria pinnatifida (Ochrophyta,Phaeophyceae) in the intertidal of North Patagonia,Argentina

Population dynamics can be influenced by physical and biological factors, particularly in stressful environments. Introduced species usually have great physiological plasticity, resulting in populations with different traits. Undaria pinnatifida, a macroalga originally described from northeast Asia, was introduced in Northern Patagonia, Argentina (San Matías Gulf) around 2010. To describe the spatio-temporal variability in population structure and morphometry of U. pinnatifida, we conducted monthly field samplings for 2 years at the intertidal area of two contrasting sites in the San Matías Gulf. Individuals of U. pinnatifida were classified by developmental stage, and their morpho-gravimetric variables were measured. In both intertidal sites juveniles were found in higher proportion during austral autumn and grew and matured during the autumn-winter months (from May onwards), and individuals senesced during early austral summer (December and January). Conversely, density and biomass were largely different between sites, and individuals showed slight morphological variability between sites. Environmental (e.g., nutrient concentration, available substrate) and biological factors (e.g., facilitation, competition) may explain the observed differences. Since there is not a macroalga with U. pinnatifida morphometrical characteristics in the intertidal environments of San Matías Gulf, studying this recent introduction gives us a better understanding of its potential ecological effects.     

Root productivity in a lowland tropical rain forest in Mexico

Fine root productivity was estimated in a lowland tropical rain forest at Los Tuxtlas (SE Mexico) and examined in relation to climatic factors. Two root diameter classes were defined (class I,<1 mm; class II, 1–3 mm). Total root productivity was estimated to 1.95 t ha^–1 year^–1, a value which is lower than those reported from other rain forest sites. Significant differences in root dry weight were found among months and between diameter classes throughout the year. Class I monthly means formed two groups: one corresponding to the months of highest precipitation, and the other to the relatively dry season. Class II monthly means also formed two groups, although these were unrelated to the regional precipitation pattern. A multiplicative regression model of productivity on precipitation was significant for both root classes when rainfall data of the previous month were used, while a linear regression model was significant only for class I roots when temperature data of two months before were used; these results suggest a delay in the effect of climatic conditions on root productivity. While the seasonal pattern of root productivity is clearly related to the annual rainfall distribution, the low total annual productivity may be related to the very high soil fertility at Los Tuxtlas.     

Ehrlich cell plasma membrane redox system is modulated through signal transduction pathways involvingcGMP and Ca2+ as second messengers

Ehrlich cell plasma membrane ferricyanide reductase activity increased in the presence of mastoparan, a generic activator of G proteins, using either whole cells or isolated plasma membrane fractions. Agents that increase intracellularcAMP also increased the rate of ferricyanide reduction by Ehrlich cells. For the first time, evidence is shown on a modulation of plasma membrane redox system bycGMP. In fact, permeant analogs ofcGMP, dibutyrylcGMP, and 8-bromo-cGMP increased the rate of ferricyanide reduction by the Ehrlich cell plasma membrane redox system. Furthermore, specific inhibition ofcGMP-phosphodiesterases by dipyridamole was also accompanied by an enhancement in the rate of ferricyanide reduction. On the other hand, treatments expected to increase cytoplasmic Ca²⁺ concentrations were accompanied by a remarkable stimulation of the reductase activity. Taking all these data together, it seems that the Ehrlich cell plasma membrane redox system is under a multiple and complex regulation by different signal transduction pathways involving G proteins, cyclic nucleotides, and Ca²⁺ ions.     

Effects of Auxin and Phenobarbital on Morphogenesis and Production of Digitoxin in Digitalis Callus

Pieces of callus obtained from seedlings of Digitalis purpureawere grown on solid Murashige-Skoog's medium supplemented with1 mg liter^–1 BA and 0.1 mg liter^–1 IAA or NAA, withor without phenobarbital (40 mg liter^–1). The replacementof the natural auxin IAA by the synthetic auxin NAA increasedcallus growth and inhibited organogenesis, whereas the additionof phenobarbital had the opposite effect. Morphometric measurementsrevealed a high ratio of vacuole to cytoplasm (v/v) in calluscells. This ratio was affected by the different treatments inthe same way as the fresh weight. The activity of mitochondrialcytochrome P450scc (the enzyme that provides the precursor,pregnenolone, for the biosynthesis of cardenolide in foxgloveplants) was detected in the relevant fraction of callus grownunder all experimental conditions, and its activity was increasedby the addition of phenobarbital. The different treatments testedincreased the cardenolide content and quantifiable amounts ofdigitoxin were detected in all callus tissues. It is of specialinterest that phenobarbital added to the culture medium increasedthe accumulation of digitoxin. The mechanism affecting the developmentand production of cardenolide in callus tissues of D. purpureaby phenobarbital and the replacement of IAA by NAA is discussed. (Received July 18, 1994; Accepted December 14, 1994)     

Distribution of nitrate reductase activity in Vicia faba: Effect of nitrate and plant genotype

The short term effect of NO₃⁻ (12 mM) on nitrate reductase (NR. EC 1.6.6.1) activity has been studied in the roots, nodules and leaves of different genotypes of Vicia faba L. at the end of vegetative growth. Root and leaf NR activity responded positively to NO₃⁻ while nodule activity, where detected, proved to he strongly inhibited. The withdraw of this NO₃⁻ from the solution consistently reduced activity in the roots and leaves but surprising, promoted a significant increase in nodule activity, which matched or surpassed that of control plants On the other hand, nodules developed in the presence of 8 mM NO₃⁻ expressed an on average 141% higher level of NR activity than did controls. This effect was observed even in nodules with negligible control activity. In any case, a naturally occurring mutant (VF17) lacking root and nodule NR activity is described. The results indicate that in V. faba. the effects of NO₃⁻ and plant genotype on NR activity depended on plant organ and time of NO₃⁻ application, hut the distribution of NO₃⁻ reduction through the plain was mainly dependent on plant genotype, and to a lesser extent on NO: supply and plant age.     

Subnucleolar location of fibrillarin and variation in its levels during the cell cycle and during differentiation of plant cells

The nucleolar protein fibrillarin has been studied in onion cells; it is detected as an Mr 37,000 protein by immunoblotting using a human autoimmune serum. Quantitative immunoelectron microscopy showed that most fibrillarin is localized in the transition zone between the fibrillar center (FC) and the dense fibrillar component (DFC) as well as in the priximal zone of the DFC, where the labeling shows a gradual decrease out-ward until it reaches insignificant levels in the distal zone of the DFC. Thus, fibrillarin is not uniformly distributed throughout the DFC of plant cells. This result supports the hypothesis that the morphologically homogeneous DFC may not be uniform in function; it is also in agreement with the hypothesized vectorial flow of ribosome biogenesis through the same compartments. Data are also presented showing that the amount of fibrillarin increase when nucleolar activity increases in G2, and probably decreases when nucleolar activity decreases during differentiation.     

Glycosylation of Acetylcholinesterase Forms in Microsomal Membranes from Normal and Dystrophic Lama2dy Mouse Muscle

Abstract: The distribution and glycosylation of acetylcholinesterase (AChE) forms in vesicles derived from sarcoplasmic reticulum of normal muscle (NMV) were investigated and compared with those from dystrophic muscle vesicles (DMV). AChE activity was similar in NMV and DMV. Most of the AChE in NMV and half in DMV were released with Triton X-100. Asymmetric (A₁₂) and globular hydrophilic and amphiphilic (G^H₄, G^A₄, G^A₂, and G^A₁) AChE species occurred in NMV and DMV, the lighter forms being predominant. The percentage of G^H₄ and G^A₄ decreased in DMV. A fraction of the AChE that could not be extracted with detergent was detached with collagenase. Most of the detergent-released A₁₂ AChE from NMV and nearly half in DMV failed to bind to Ricinus communis agglutinin (RCA-I). Conversely, the collagenase-detached isoforms bound to RCA, revealing that asymmetric AChE associated with internal membranes or basal lamina differed in glycosylation. Moreover, nearly half of G^A₄ AChE in DMV and a few in NMV bound to RCA. Most of the RCA-unreactive G^A₄ forms in NMV come from sarcolemma. The results indicate that dystrophy induces minor changes in the distribution and glycosylation of AChE forms in internal membranes of muscle.