Impact of Atmospheric Sulfur Deposition on Sulfur Metabolism in Plants: H2S as Sulfur Source for Sulfur Deprived Brassica oleracea L.

Brassica oleracea L. was rather insensitive to atmospheric H₂S: growth was only negatively affected at ≥0.4 μl I^?1. Shoots formed a sink for H₂S and the uptake rate showed saturation kinetics with respect to the atmospheric concentration. The H₂S uptake rate was high in comparison with other species, which may reflect the high sulfur need of Brassica. The net uptake of sulfate by roots of hydroponically grown plants was substantially reduced after one week of exposure to 0.25 μl l^?1 H₂S, indicating that plants switched in part from sulfate to H₂S as sulfur source for plant growth. Plants were sulfur deficient after two weeks of sulfur deprivation, illustrated by reduced growth, which was more pronounced for shoots than for roots, and in enhanced shoot dry matter content. The latter could for the greater part be attributed to enhanced levels of soluble sugars and starch. Sulfur deficiency was further characterized by a low pigment content, extremely low levels of sulfate and water-soluble non-protein thiols, and by enhanced levels of nitrate and free amino acids, particularly in the shoots. Furthermore, sulfur deficient plants contained a lower total lipid content in shoots, whereas its content in roots was unaffected. The level of sulfolipids was decreased in both roots and shoots. When sulfur deprived plants were exposed to 0.25 μl I^?1 H₂S for one week, all sulfur deficiency symptoms were abolished and growth was restored. Furthermore, plants were able to grow with 0.4 μl I^?1 H₂S as the sole sulfur source. Water-soluble non-protein thiol content was enhanced in both shoots and roots of H₂S exposed plants, irrespective of the sulfate supply to the roots, whereas plants grown with H₂S as sole sulfur source contained very low sulfate levels. The interaction between atmospheric and pedospheric sulfur nutrition in plants is discussed.     

Photoperiodic Control of Developmental Diapause in Nymphs of Prostriate Ixodid Ticks (Acari: Ixodidae)

Extrinsic control of developmental diapause in nymphs of prostriate ticks of the subgenus Ixodes sensu stricto (Ixodes ricinus and Ixodes persulcatus from Eurasia and Ixodes scapularis from North America) appears to be based on a complex two-step photoperiodic reaction of a short-day/long-day type. Diapause control in the subgenus Afrixodes (the South African tick Ixodes rubicundus) appears to be based on a simple long-day reaction. The option between non-diapause development and diapausing arrest in engorged nymphs is determined by both pre- and post-feeding photoperiodic regimes. Consequently diapausing arrest in engorged nymphs of Ixodes sensu stricto can be induced either by a short-day (after their engorgement) or by a long-day regime (in unfed nymphs), while active, non-diapause development is possible only when the short-day pre-feeding regime is followed by a long-day post-feeding regime. The photoperiodic response in I. (Afrixodes) rubicundus nymphs seems to be of the long-day type both before and after feeding. Consequently this non-diapause development is enabled by a long-day regime, while diapause is induced by a short-day regime of exposure. Nevertheless, there are some indications that the control of nymphal diapause in the latter species is also of a complex nature. This revised version was published online in July 2006 with corrections to the Cover Date.     

Functional and structural characterization of a synthetic peptide representing the N-terminal domain of prokaryotic pyruvate dehydrogenase

A synthetic peptide (Nterm-E1p) is used to characterize the structure and function of the N-terminal region (amino acid residues 4-45) of the pyruvate dehydrogenase component (E1p) from the pyruvate dehydrogenase multienzyme complex (PDHC) from Azotobacter vinelandii. Activity and binding studies established that Nterm-E1p specifically competes with E1p for binding to the dihydrolipoyl transacetylase component (E2p) of PDHC. Moreover, the experiments show that the N-terminal region of E1p forms an independent folding domain that functions as a binding domain. CD measurements, two-dimensional (2D) (1)H NMR analysis, and secondary structure prediction all indicate that Nterm-E1p has a high alpha-helical content. Here a structural model of the N-terminal domain is proposed. The peptide is present in two conformations, the population of which depends on the sample conditions. The conformations are designated "unfolded" at pH > or =6 and "folded" at pH <5. The 2D (1)H TOCSY spectrum of a mixture of folded and unfolded Nterm-E1p shows exchange cross-peaks that "link" the folded and unfolded state of Nterm-E1p. The rate of exchange between the two species is in the range of 0.5-5 s(-1). Sharp resonances in the NMR spectra of wild-type E1p demonstrate that this 200 kDa enzyme contains highly flexible regions. The observed dynamic character of E1p and of Nterm-E1p is likely required for the binding of the E1p dimer to the two different binding sites on E2p. Moreover, the flexibility might be essential in sustaining the allosteric properties of the enzyme bound in the complex.     

The toolbox of vesicle sidedness determination

Vesicles prepared from cellular plasma membranes are widely used in science for different purposes. The outer membrane leaflet differs from the inner membrane leaflet of the vesicle, and during vesicle preparation procedures two types of vesicles will be generated: right-side-out vesicles, of which the outer leaflet is topologically equivalent to the outer monolayer of the cellular plasma membrane, and inside-out vesicles. Because two populations of vesicles exist, sidedness information of the vesicle preparation is indispensable. This note focuses on the ins and outs of sidedness determination of vesicles and compares various methodologies used to establish this ratio.     

Separation of Antioxidant-Rich Alternanthera Sessilis Red Extracts by Sephadex LH-20 and Identification of Polyphenols Using HPLC-QToF-MS/MS

This study aimed to fractionate Alternanthera sessilis Red (ASR) crude extracts and determine their antioxidant activities as well as the related active components in the whole plant. ASR was extracted with water and ethanol, and further separated using a Sephadex LH-20 column. Following the assessments of the polyphenolic contents and antioxidant activities of crude extracts (H₂O_ASR and EtOH_ASR) and fractions, a HPLC-QToF analysis was performed on the crude extracts and selected fractions (H₂O_ASR FII and EtOH_ASR FII). Three water fractions (H₂O_ASR FI, FII and FIII) and four ethanolic fractions (EtOH_ASR FI, FII, FIII and FIV) were derived from their crude extracts, respectively. EtOH_ASR FII exhibited the greatest total phenolic content (120.41 mg GAE/g fraction), total flavonoid content (223.07 mg RE/g fraction), and antioxidant activities (DPPH IC₅₀=159.43 μg/mL; FRAP=1.93 mmol Fe²⁺/g fraction; TEAC=0.90 mmol TE/g fraction). Correlation analysis showed significant (p<0.01) positive correlations between both TPC (r=0.748–0.970) and TFC (r=0.686–0.949) with antioxidant activities in the crude extracts and fractions. Flavonoids were the major compounds in the four selected samples tentatively identified using HPLC-QToF-MS/MS, with the highest number of 30 polyphenol compounds detected in the most active fraction, EtOH_ASR FII.     

Body fluid responses of heat-tolerant and intolerant men to work in a hot wet environment.

Acclimatization to heat before proceeding underground is a requirement for each South African mine laborer. Certain individuals among this large population cannot be acclimatized to heat (33.3 degrees C db, 31.7 degrees C wb) and are classified as heat intolerant. In this study certain body fluid responses to heat and work were compared between a group of 19 heat-tolerant (HT) and of 15 heat-intolerant (HI) subjects. To the factors known to affect heat tolerance such as age, weight, and oxygen consumption must now be added differences in body fluid responses. The HI group of subjects failed to hemodilute to the same degree as the HT group though working at the same relative work loads (30% and 50% VO2 max). As the 4-h work period (33.3 degrees C db, 31.7 degrees C wb) continued, the HI group did not maintain hemodilution in spite of the lower absolute work loads, sweat rates, and water deficits suffered by this group. From analysis of blood constituent changes it was suggested that the reason for the differences noted in body fluid dynamics concerned plasma protein equilibrium across capillary walls as well as the protein population of interstitial spaces.     

Human TRBP and PACT directly interact with each other and associate with dicer to facilitate the production of small interfering RNA

Mammalian Dicer interacts with double-stranded RNA-binding protein TRBP or PACT to mediate RNA interference and micro-RNA processing. TRBP and PACT are structurally related but exert opposite regulatory activities on PKR. It is not understood whether TRBP and PACT are simultaneously required for Dicer. Here we show that TRBP directly interacts with PACT in vitro and in mammalian cells. TRBP and PACT form a triple complex with Dicer and facilitate the production of small interfering RNA (siRNA) by Dicer. Knockdown of both TRBP and PACT in cultured cells leads to significant inhibition of gene silencing mediated by short hairpin RNA but not by siRNA, suggesting that TRBP and PACT function primarily at the step of siRNA production. Taken together, these findings indicate that human TRBP and PACT directly interact with each other and associate with Dicer to stimulate the cleavage of double-stranded or short hairpin RNA to siRNA. Our work significantly alters the current model for the assembly and function of the Dicer-containing complex that generates siRNA and micro-RNA in human.     

Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging

One approach to super-resolution fluorescence imaging uses sequential activation and localization of individual fluorophores to achieve high spatial resolution. Essential to this technique is the choice of fluorescent probes; the properties of the probes, including photons per switching event, on-off duty cycle, photostability and number of switching cycles, largely dictate the quality of super-resolution images. Although many probes have been reported, a systematic characterization of the properties of these probes and their impact on super-resolution image quality has been described in only a few cases. Here we quantitatively characterized the switching properties of 26 organic dyes and directly related these properties to the quality of super-resolution images. This analysis provides guidelines for characterization of super-resolution probes and a resource for selecting probes based on performance. Our evaluation identified several photoswitchable dyes with good to excellent performance in four independent spectral ranges, with which we demonstrated low-cross-talk, four-color super-resolution imaging.     

Membrane topology of the lactococcal bacteriocin ATP-binding cassette transporter protein LcnC. Involvement of LcnC in lactococcin a maturation

Many non-lantibiotic bacteriocins of lactic acid bacteria are produced as precursors with N-terminal leader peptides different from those present in preproteins exported by the general sec-dependent (type II) secretion pathway. These bacteriocins utilize a dedicated (type I) secretion system for externalization. The secretion apparatus for the lactococcins A, B, and M/N (LcnA, B, and M/N) from Lactococcus lactis is composed of the two membrane proteins LcnC and LcnD. LcnC belongs to the ATP-binding cassette transporters, whereas LcnD is a protein with similarities to other accessory proteins of type I secretion systems. This paper shows that the N-terminal part of LcnC is involved in the processing of the precursor of LcnA. By making translational fusions of LcnC to the reporter proteins beta-galactosidase (LacZ) and alkaline phosphatase (PhoA*), it was shown that both the N- and C-terminal parts of LcnC are located in the cytoplasm. As the N terminus of LcnC is required for LcnA maturation and is localized in the cytoplasm, we conclude that the processing of the bacteriocin LcnA to its mature form takes place at the cytosolic side of the cytoplasmic membrane.     

Do mitochondria make nitric oxide? no?

Several papers have claimed that mitochondria contain nitric oxide synthase (NOS) and make nitric oxide (NO*) in amounts sufficient to affect mitochondrial respiration. However, we found that the addition of L-arginine or the NOS inhibitor L-NMMA to intact rat liver mitochondria did not have any effect on the respiratory rate in both State 3 and State 4. We did not detect mitochondrial NO* production by the oxymyoglobin oxidation assay, or electrochemically using an NO* electrode. An apparent NO* production detected by the Griess assay was identified as an artifact. NO* generated by eNOS added to the mitochondria could easily be detected, although succinate-supplemented mitochondria appeared to consume NO*. Our data show that NO* production by normal rat liver mitochondria cannot be detected in our laboratory, even though the levels of production claimed in the literature should easily have been measured by the techniques used. The implications for the putative mitochondrial NOS are discussed.