Study of a time lag in the assay of Escherichia coli membrane-bound dehydrogenases based on tetrazolium salt reduction

The Escherichia coli membrane-bound D-lactate dehydrogenase and succinate dehydrogenase were assayed on the basis of the phenazine methosulfate- (PMS-) mediated reduction of the tetrazolium salt, MTT. An initial slower phase (lag) in the time-course of the reaction was observed and analyzed. The results were as follows. (1) The time lag in the assay of the D-lactate dehydrogenase was eliminated by preincubating the membranes with PMS plus D-lactate, with PMS plus succinate, or with PMS plus NADH (conditions which implicated PMS reduction). (2) When the D-lactate dehydrogenase was assayed by another method based on the measurement of the pyruvate formed, neither was a time lag observed nor was the enzyme activity affected by membrane preincubation with PMS plus D-lactate. (3) Although the superoxide radical was involved in MTT reduction, this radical seemed not to participate in the generation of the time lag. (4) Membranes whose D-lactate dehydrogenase activity had previously been destroyed by heating at 80 degrees C for 1 min, were able to prolong the time lag in MTT reduction when added to the assay medium for the D-lactate dehydrogenase from untreated membranes, whereas membranes previously heated at 100 degrees C instead of 80 degrees C did not have this effect. It was concluded that the E. coli membranes interfered in the dehydrogenase assay based on the PMS-mediated reduction of MTT. The time lag was interpreted as a period during which the interfering substance reacted with reduced PMS inhibiting the reduction of MTT.     

Cryptic species and colonization processes in Ophryotrocha (Annelida,Dorvilleidae) inhabiting vertebrate remains in the shallow‐water Mediterranean

Over the past several years, there has been growing interest in how bones of decaying mammals are colonized in the marine seabed. One of the most common opportunistic taxa occurring worldwide on bones is dorvilleid polychaetes of the genus Ophryotrocha. In a recent study in the Mediterranean, Ophryotrocha puerilis and Ophryotrocha alborana were two of the most abundant species occurring in experimentally deployed bones. These species have direct development and this makes them a suitable model to study the mechanisms and processes allowing organisms lacking a dispersive larval phase to colonize new substrates. Here, we address the colonization processes at the molecular level for populations of O. puerilis and O. alborana on experimentally deployed mammal bones in the shallow‐water Mediterranean collected over a year at 3‐month intervals. High genetic distances between some of the O. puerilis organisms collected indicated the occurrence of at least two cryptic sibling species (O. puerilis ‘Shallow’ and O. puerilis ‘Deep’) apart from O. puerilis sensu stricto. This was corroborated with phylogenetic analyses using an alignment of three concatenated genes (COI, 16S, H3) and with species delimitation analyses using COI. The haplotype network inferred from COI sequences for O. puerilis ‘Shallow’ showed a few common haplotypes shared between the two trimesters analysed and several other less represented haplotypes only present in one trimester. Thus, colonization of these experimental bones may have been achieved by a few organisms that arrived to the bones and were able to reseed, and by several individuals arriving to the experimental bones and not persisting across time. In contrast, the haplotype network for O. alborana revealed that none of the haplotypes present in three different trimesters were shared, suggesting that the populations arriving at the bones during each trimester were totally replaced by new individuals during the subsequent trimesters. Our study suggests that different species of shallow‐water Ophryotrocha occurring in the Mediterranean may have different patterns of substrate colonization despite sharing similar life histories.     

ABCG transporters export cutin precursors for the formation of the plant cuticle

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Assessment of a 1H high-resolution magic angle spinning NMR spectroscopy procedure for free sugars quantification in intact plant tissue

In most plants, sucrose is the primary product of photosynthesis, the transport form of assimilated carbon, and also one of the main factors determining sweetness in fresh fruits. Traditional methods for sugar quantification (mainly sucrose, glucose and fructose) require obtaining crude plant extracts, which sometimes involve substantial sample manipulation, making the process time-consuming and increasing the risk of sample degradation. Here, we describe and validate a fast method to determine sugar content in intact plant tissue by using high-resolution magic angle spinning nuclear magnetic resonance spectroscopy (HR-MAS NMR). The HR-MAS NMR method was used for quantifying sucrose, glucose and fructose in mesocarp tissues from melon fruits (Cucumis melo var. reticulatus and Cucumis melo var. cantalupensis). The resulting sugar content varied among individual melons, ranging from 1.4 to 7.3 g of sucrose, 0.4–2.5 g of glucose; and 0.73–2.83 g of fructose (values per 100 g fw). These values were in agreement with those described in the literature for melon fruit tissue, and no significant differences were found when comparing them with those obtained using the traditional, enzymatic procedure, on melon tissue extracts. The HR-MAS NMR method offers a fast (usually <30 min) and sensitive method for sugar quantification in intact plant tissues, it requires a small amount of tissue (typically 50 mg fw) and avoids the interferences and risks associated with obtaining plant extracts. Furthermore, this method might also allow the quantification of additional metabolites detectable in the plant tissue NMR spectrum.     

Release of bound immunoglobulin from SSPE brain by acid elution.

SSPE brain homogenate extracted at pH 7.4 yields immunoglobulin with a 4- to 5-fold greater hemagglutination inhibition activity per microgram of IgG than serum from the same patient. Serial washing of the homogenate results in a low level steady-state release of IgG. Elution of the washed sediment with pH 2.5, 0.1 M glycine buffer results in a 2- to 3-fold increase in recovery of hemagglutination inhibition activity with a greater hemagglutination inhibition activity per milligram of IgG than the IgG recovered by phosphate-saline extraction at pH 7.4.     

Evidence for a nigro-pulvinar-lateralis posterior complex projection in the cat using horseradish peroxidase neuronal retrograde technique

The possible existence of a direct projection from the substantia nigra to the pulvinar-lateral posterior complex (Pul-LP) was investigated in the cat by using the horseradish peroxidase technique. In particular horseradish peroxidase was injected in the Pul-LP of 8 animals, either unilaterally or bilaterally. Tissue sections obtained from the cat's brain 24-48 hrs. after injection were prepared according to Mesulam's method as slightly modified by the authors. Retrogradelly labelled neurons were observed in substantia nigra pars lateralis and reliculata ipsilaterally to the injected pulvinar-lateral posterior complex. A small number of labelled cells were also found in the contralateral substantia nigra. These findings demonstrate the existence of a close connection between two system which are involved in turning behavior: the nigrostriatal and the pulvinar-lateral posterior complex-superior colliculus.     

Selection of phosphate-solubilizing diazotrophic Herbaspirillum and Burkholderia strains and their effect on rice crop yield and nutrient uptake

Background and Aims

Plant growth-promoting bacteria, mainly diazotrophs and phosphate solubilizers, can reduce the use of chemical fertilizers for rice crops. Here, diazotrophic bacteria isolated from rice were screened for their ability to solubilize inorganic P (P_i) in vitro and in association with rice plants cultivated in pots.

Methods

Forty-nine isolates were tested for the ability to solubilize P_i on NBRIP and GL agar plate media and seven selected strains were further evaluated in NBRIP liquid medium. Three of these strains were inoculated in rice plants grown in soil pots containing ¹⁵N-labeled fertilizer and two sources of P: tricalcium phosphate (TCP) or simple superphosphate (SSP). The dry matter, yield, N, P, and the ¹⁵N content accumulated in plant tissues were measured at 135 days after planting.

Results

Seven strains belonging to the genera Herbaspirillum and Burkholderia formed a halo of solubilized P_i on agar plates. The Burkholderia strains showed peak soluble P (around 200 mg P L^?1) on the fifth day when grown in NBRIP liquid medium for 14 days. Inoculation of Herbaspirillum strains (H18, ZA15) and a Burkholderia vietaminensis strain (AR114) increased rice grain yield from 33 to 47 % with TCP and 18 to 44 % with TSS, respectively. The bacterial inoculation led to enhanced N-use efficiency of the ¹⁵N-labeled fertilizer.

Conclusion

These results suggest that the selection and use of P-solubilizing diazotrophic bacteria are a good strategy to promote P solubilization and/or N use efficiency in rice plants.     

Biogeographical Patterns and Phenological Changes in Lapiedra martinezii Lag. Related to Its Alkaloid Diversity

The aim of this work was to investigate the alkaloid patterns of Lapiedra martinezii and their relation to biogeography and phenology focused in a phylogenetic comparison. Plants from 14 populations of L. martinezii, covering almost its entire distribution area, were subjected to morphological, ecological, and phytochemical analysis. Experiments for different alkaloid‐type content are proposed as a new tool for analysis of plant distribution. Several plants were transplanted for weekly observation of their phenological changes, and alkaloids from different plant organs were extracted, listed, and compared. The alkaloid pattern of L. martinezii comprises 49 compounds of homolycorine, lycorine, tazettine, haemantamine, and narciclasine types. The populations located in the north and south margins of the distribution area displayed alkaloid patterns different from those of the central area. Changes in these patterns during their phenological cycle may be related to a better defence for plant reproduction. L. martinezii is an old relict plant, and it has maintained some of the more primitive morphological features and alkaloid profiles of the Mediterranean Amaryllidaceae. The variations in alkaloid content observed could be interpreted in a phylogenetic sense, and those found in their phenological changes, in an adaptive one.     

Rab11‐Family of Interacting Protein 2 associates with chlamydial inclusions through its Rab‐binding domain and promotes bacterial multiplication

Chlamydia trachomatis, an obligate intracellular pathogen, survives within host cells in a special compartment named ‘inclusion’ and takes advantage of host vesicular transport pathways for its growth and replication. Rab GTPases are key regulatory proteins of intracellular trafficking. Several Rabs, among them Rab11 and Rab14, are implicated in chlamydial development. FIP2, a member of the Rab11‐Family of Interacting Proteins, presents at the C‐terminus a Rab‐binding domain that interacts with both Rab11 and Rab14. In this study, we determined and characterized the recruitment of endogenous and GFP‐tagged FIP2 to the chlamydial inclusions. The recruitment of FIP2 is specific since other members of the Rab11‐Family of Interacting Proteins do not associate with the chlamydial inclusions. The Rab‐binding domain of FIP2 is essential for its association. Our results indicate that FIP2 binds to Rab11 at the chlamydial inclusion membrane through its Rab‐binding domain. The presence of FIP2 at the chlamydial inclusion favours the recruitment of Rab14. Furthermore, our results show that FIP2 promotes inclusion development and bacterial replication. In agreement, the silencing of FIP2 decreases the bacterial progeny. C. trachomatis likely recruits FIP2 to hijack host intracellular trafficking to redirect vesicles full of nutrients towards the inclusion.     

Clinanthus microstephium,an Amaryllidaceae Species with Cholinesterase Inhibitor Alkaloids: Structure−Activity Analysis of Haemanthamine Skeleton Derivatives

Plants of the Amaryllidaceae family are well‐known (not only) for their ornamental value but also for the alkaloids that they produce. In this report, the first phytochemical study of Clinanthus genus was carried out. The chemical composition of alkaloid fractions from Clinanthus microstephium was analyzed by GC/MS and NMR. Seven known compounds belonging to three structural types of Amaryllidaceae alkaloids were identified. An epimeric mixture of a haemanthamine‐type compound (6‐hydroxymaritidine) was tested as an inhibitor against acetyl‐ and butyrylcholinesterase enzymes (AChE and BChE, respectively), two enzymes relevant in the treatment of Alzheimer's disease, with good results. Structure–activity relationships through molecular docking studies with this alkaloid and other structurally related compounds were discussed.