Uptake of Rhodnius heme-binding protein (RHBP) by the ovary of Rhodnius prolixus

The uptake of RHBP (Rhodnius heme-binding protein) by the ovaries of Rhodnius prolixus was characterized. RHBP purified from oocyte was labeled with ¹²⁵I and used to study the process of uptake by the ovary in vivo and in vitro. After injection, the [¹²⁵I]RHBP was readily removed from the hemolymph and accumulated especially in the ovary. The capacity of the ovary to take up [¹²⁵I]RHBP from the hemolymph varied during the days following blood meal. It increased up to day 2, remained stable until day 5, and then decreased up to the end of oogenesis. In vitro, the uptake of [¹²⁵I]RHBP was linear at least up to 60 min. The uptake was dependent on [¹²⁵I]RHBP concentration and showed to be a saturable process. The addition of a molar excess of non-related proteins such as Vitellin (Vt), Lipophorin (Lp), and Bovine Serum Albumin (BSA) did not reduce [¹²⁵I]RHBP uptake. Using immunogold technique the RHBP was localized at the microvilli, coated pits, and yolk granules. The main yolk protein, Vt, did not compete with RHBP for the uptake. Thus, it is discussed here that they bind to independent binding sites of the oocytes, and are directed later on to the same compartment. The need of both proteins for the completion of mature oocyte was verified in vivo. The reduction of heme-RHBP in the hemolymph, by changing the diet, decreased the number of eggs laid. Increasing the concentration of heme-RHBP in the hemolymph, the number of eggs produced increased in a dose dependent manner. In vitro, both apo-RHBP and heme-RHBP can be taken up by the oocyte. Since the mature oocyte contains only heme-saturated RHBP, the possible fate of apo-RHBP is also discussed. Arch. Insect Biochem. Physiol. 39:133–143, 1998. © 1998 Wiley-Liss, Inc.     

Lipoic acid entrains the hepatic circadian clock and lipid metabolic proteins that have been desynchronized with advanced age

It is well established that lipid metabolism is controlled, in part, by circadian clocks. However, circadian clocks lose temporal precision with age and correlates with elevated incidence in dyslipidemia and metabolic syndrome in older adults. Because our lab has shown that lipoic acid (LA) improves lipid homeostasis in aged animals, we hypothesized that LA affects the circadian clock to achieve these results. We fed 24 month old male F344 rats a diet supplemented with 0.2% (w/w) LA for 2 weeks prior to sacrifice and quantified hepatic circadian clock protein levels and clock-controlled lipid metabolic enzymes. LA treatment caused a significant phase-shift in the expression patterns of the circadian clock proteins Period (Per) 2, Brain and Muscle Arnt-Like1 (BMAL1), and Reverse Erythroblastosis virus (Rev-erb) β without altering the amplitude of protein levels during the light phase of the day. LA also significantly altered the oscillatory patterns of clock-controlled proteins associated with lipid metabolism. The level of peroxisome proliferator-activated receptor (PPAR) α was significantly increased and acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) were both significantly reduced, suggesting that the LA-supplemented aged animals are in a catabolic state. We conclude that LA remediates some of the dyslipidemic processes associated with advanced age, and this mechanism may be at least partially through entrainment of circadian clocks.     

Physiologic and metagenomic attributes of the rhodoliths forming the largest CaCO3 bed in the South Atlantic Ocean

Rhodoliths are free-living coralline algae (Rhodophyta, Corallinales) that are ecologically important for the functioning of marine environments. They form extensive beds distributed worldwide, providing a habitat and nursery for benthic organisms and space for fisheries, and are an important source of calcium carbonate. The Abrolhos Bank, off eastern Brazil, harbors the world''s largest continuous rhodolith bed (of ∼21 000 km²) and has one of the largest marine CaCO₃ deposits (producing 25 megatons of CaCO₃ per year). Nevertheless, there is a lack of information about the microbial diversity, photosynthetic potential and ecological interactions within the rhodolith holobiont. Herein, we performed an ecophysiologic and metagenomic analysis of the Abrolhos rhodoliths to understand their microbial composition and functional components. Rhodoliths contained a specific microbiome that displayed a significant enrichment in aerobic ammonia-oxidizing betaproteobacteria and dissimilative sulfate-reducing deltaproteobacteria. We also observed a significant contribution of bacterial guilds (that is, photolithoautotrophs, anaerobic heterotrophs, sulfide oxidizers, anoxygenic phototrophs and methanogens) in the rhodolith metagenome, suggested to have important roles in biomineralization. The increased hits in aromatic compounds, fatty acid and secondary metabolism subsystems hint at an important chemically mediated interaction in which a functional job partition among eukaryal, archaeal and bacterial groups allows the rhodolith holobiont to thrive in the global ocean. High rates of photosynthesis were measured for Abrolhos rhodoliths (52.16 μmol carbon m⁻²s⁻¹), allowing the entire Abrolhos rhodolith bed to produce 5.65 × 10⁵ tons C per day. This estimate illustrates the great importance of the Abrolhos rhodolith beds for dissolved carbon production in the South Atlantic Ocean.     

<i>In Vitro</i>-Propagation of <i>Agave tequilana</i> Weber cv. azul in a Temporary Immersion System

In Mexico, there is a need to produce large quantities of plantlets for the establishment and replanting of blue (cv. azul) agave production areas. Most of these plots are within the origin denomination area (DOT, Spanish acronym) of the distilled product of this plant, known as tequila. The objective of this study was to develop an in vitro-propagation protocol for Agave tequilana Weber cv. azul using segmented stems in both: solid and liquid media. A disinfection and in vitro technique were developed to obtain shoots, through plantlets collected in commercial plots, which attained 100% surface-disinfection and budding rate. At the multiplication stage, the effects of 6-Benzylaminopurine (BA) (0.0, 4.4 and 13.2 μM) and kinetin (0.0, 9.4, 18.8 and 37.6 μM) were evaluated on lateral-shoot production of segmented sagittal stems. These were cultivated on Murashige & Skoog (MS) medium, with the addition of 3.0% sucrose and 8 g L⁻¹ agar. It was observed that BA and kinetin increased the number of shoots per explant, obtaining up to 18 and 26, respectively. Furthermore, it was found that just the sagittal segmentation of explants increased axillary budding. On the other hand, segmented-stem bases were grown in MS liquid medium with 3.0% sucrose, inside a RITA® system, programmed by a 5 min immersion step with a frequency of every 4 h. The effect of Indole−3-Acetic acid (IAA) (0.57, 2.9, 5.7 μM) was evaluated, while maintaining a concentration of BA (13.2 μM). It was observed that the greatest concentration of IAA led to the formation of more than 20 buds per explant. These results offer a new methodology to increase the efficiency of A. tequilana Weber cv. azul-in vitro multiplication by sagittal segmentation of stems and the addition of BA and/or IAA.     

The cloning, expression, purification, characterization and modeled structure of Caulobacter crescentus β-Xylosidase I

The xynB1 gene (CCNA 01040) of Caulobacter crescentus that encodes a bifunctional enzyme containing the conserved β-Xylosidase and α-L: -Arabinofuranosidase (β-Xyl I-α-L: -Ara) domains was amplified by PCR and cloned into the vector pJet1.2Blunt. The xynB1 gene was subcloned into the vector pPROEX-hta that produces a histidine-fused translation product. The overexpression of recombinant β-Xyl I-α-L: -Ara was induced with IPTG in BL21 (DE3) and the resulting intracellular protein was purified with pre-packaged nickel-Sepharose columns. The recombinant β-Xyl I-α-L: -Ara exhibited a specific β-Xylosidase I activity of 1.25?U?mg(-1) to oNPX and a specific α-L: -Arabinofuranosidase activity of 0.47?U?mg(-1) to pNPA. The predominant activity of the recombinant enzyme was its β-Xylosidase I activity, and the enzymatic characterization was focused on it. The β-Xylosidase I activity was high over the pH range 3-10, with maximal activity at pH 6. The enzyme activity was optimal at 45?°C, and a high degree of stability was verified over 240?min at this temperature. Moreover, β-Xylosidase activity was inhibited in the presence of the metals Zn(2+) and Cu(2+), and the enzyme exhibited K(M) and V(Max) values of 2.89?±?0.13?mM and 1.4?±?0.04?μM?min(-1) to oNPX, respectively. The modeled structure of β-xylosidase I showed that its active site is highly conserved compared with other structures of the GH43 family. The increase in the number of contact residues responsible for maintaining the dimeric structure indicates that this dimer is more stable than the tetramer form.     

Changes in membrane lipids and carotenoids during light acclimation in a marine cyanobacterium Synechococcus sp.

Time course of carotenoid and membrane lipid variation during high light (HL) acclimation (about 85 meu mol m-2 s-1), after transfer from low light (LL) (5-10 meu mol m-2 s-1), was determined in a marine Synechococcus strain. Highperformance liquid chromatography (HPLC) coupled to diode array detector (DAD) or electrospray ionization mass spectrometry (ESI-MS) was used for compound separation and detection. Myxoxanthophyll rose within a time interval of 8 h to 24 h after the onset of exposure to HL. Beta -carotene content started to decrease after 4 h of the onset of exposure to HL. Zeaxanthin content rose with exposure to HL, but it was only significant after 24 h of exposure. Carotenoid changes are in agreement with a coordinated activity of the enzymes of the myxoxanthophyll biosynthetic pathway, with no rate-limiting intermediate steps. Lipid analysis showed all species with a C18:3/C16:0 composition increased their content, the changes of PG (18:3/16:0) and MGDG(18:3/16:0) being primarily significant. Major lipid changes were also found to occur within 24 h. These changes might suggest reduction and reorganization of the thylakoid membrane structure. Hypotheses are also drawn on the role played by lipid molecule shape and their possible effect in membrane fluidity and protein accommodation.