Plant gas exchange at high wind speeds

High altitude Rhododendron ferrugineum L. and Pinus cembra L. seedlings were exposed to winds at 15 meters per second for 24-hour periods. Wind-sensitive stomata of Rhododendron seedlings immediately initiated a closing response which resulted in decreased photosynthesis and an even greater reduction in transpiration. Stomatal aperture and transpiration rates of P. cembra were only slightly reduced by high speed winds. However, photosynthesis was substantially reduced because of changes in needle display to available irradiation.     

Effect of water immersion on cardiopulmonary physiology at high gravity (+Gz)

We compared the cardiopulmonary physiology of eight subjects exposed to 1, 2, and 3 Gz during immersion (35 degrees C) to the heart level with control dry rides. Immersion should almost cancel the effects of gravity on systemic circulation and should leave the lung alone to gravitational influence. During steady-state breathing we measured ventilation, O2 consumption (VO2), CO2 production, end-tidal PCO2 (PACO2), and heart frequency (fH). Using CO2 rebreathing techniques, we measured cardiac output, functional residual capacity, equivalent lung tissue volume, and mixed venous O2 content, and we calculated arterial PCO2 (PaCO2). As Gz increased, ventilation, fH, and VO2 rose markedly, and PACO2 and PaCO2 decreased greatly in dry ride, but during immersion these variables changed very little in the same direction. Functional residual capacity was lower during immersion and decreased in both the dry and immersed states as Gz increased, probably reflecting closure effects. Cardiac output decreased as Gz increased in dry rides and was elevated and unaffected by Gz during immersion. We conclude that most of the changes we observed during acceleration are due to the effect on the systemic circulation, rather than to the effect on the lung itself.     

Development ofTrichogramma australicum (Hym.: Trichogrammatidae) at low and high population density in artificial diet

We compared the development ofTrichogramma australicum Girault in artificial-diet filled artificial “eggs” containing 10–15 (low density) or 35–50 (high density) immature parasitoids. The size of low- and high-densityT. australicum eggs at 1, 15, 30 and 36 h was not significantly different. Also, incubation time of low- and high-density eggs in artificial diet was similar. Appearance of urate bodies in larvae developing low and high density occurred at the same time (100–108 h). Size of immatureT. australicum in low density significantly increased after the urate bodies had appeared. Black deposits appeared on the surface ofT. australicum prepupae soon after all diet had been consumed (at 108–120 and 144–168 h in low and high density, respectively). We concluded that the appearance of the black deposits was an indication of the onset of the prepupal stage. The pupal size was significantly larger at low density than high density. Mortality of prepupae and pupae was significantly higher at low density than at high density. Proportion of larvae at high density developed to abnormal and normal wasps. Wasps did not emerge from larvae reared at low density.     

Structural transition from the random coil to quadruplex of AG(3)(T(2)AG(3))(3) induced by Zn(2+)

Structures of G-quadruplex DNAs can be typically stabilized by monovalent cations such as K(+), Na(+). Some divalent and trivalent cations, such as Sr(2+), Pb(2+), Tb(3+) and Eu(3+), can also induce the formation of G-quadruplex DNA. Here we show that Zn(2+) can induce the human telomeric sequence AG(3)(T(2)AG(3))(3) to fold the G-quadruplex structure by UV absorbance difference spectra and circular dichroism (CD) spectroscopy. At micromolar concentrations, the Zn(2+)-induced changes in the UV absorbance difference spectra and CD spectra are the characteristics of antiparallel G-quadruplexes although the long wavelength CD maximum is around 285 nm rather than the typical value of 295 nm. The binding stoichometry of Zn(2+) per one AG(3)(T(2)AG(3))(3) molecule is four. To our knowledge, the structural transition of human telomeric sequence induced by Zn(2+) was observed for the first time.     

PLEKHM1: Adapting to life at the lysosome

The endosomal system and autophagy are 2 intertwined pathways that share a number of common protein factors as well as a final destination, the lysosome. Identification of adaptor platforms that can link both pathways are of particular importance, as they serve as common nodes that can coordinate the different trafficking arms of the endolysosomal system. Using a mass spectrometry approach to identify interaction partners of active (GTP-bound) RAB7, the late endosome/lysosome GTPase, and yeast 2-hybrid screening to identify LC3/GABARAP interaction partners we discovered the multivalent adaptor protein PLEKHM1. We discovered a highly conserved LC3-interaction region (LIR) between 2 PH domains of PLEKHM1 that mediated direct binding to all LC3/GABARAP family members. Subsequent mass spectrometry analysis of PLEKHM1 precipitated from cells revealed the HOPS (homotypic fusion and protein sorting) complex as a prominent interaction partner. Functionally, depletion of PLEKHM1, HOPS, or RAB7 results in decreased autophagosome-lysosome fusion. In Plekhm1 knockout (KO) mouse embryonic fibroblasts (MEFs) we observed increased lipidated LC3B, decreased colocalization between LC3B and LAMP1 under amino acid starvation conditions and decreased autolysosome formation. Finally, PLEKHM1 binding to LC3-positive autophagosomes was also essential for selective autophagy pathways, as shown by clearance of puromycin-aggregates, in a PLEKHM1-LIR-dependent manner. Overall, we have identified PLEKHM1 as an endolysosomal adaptor platform that acts as a central hub to integrate endocytic and autophagic pathways at the lysosome.PLEKHM1 (pleckstrin homology domain containing, family M [with RUN domain] member 1) is a ubiquitously expressed protein involved in the regulation of osteoclast function and bone resorption. Recently, it was also described in the context of negatively regulating the endocytic pathway but not autophagy. However, in our recent studies, we show that PLEKHM1 positively regulates the terminal stages of both endocytic and autophagy pathways through direct interaction between PLEKHM1, RAB7, the HOPS complex, and mammalian Atg8 proteins (Fig. 1A). In addition, the PLEKHM1-RAB7-HOPS complex is a direct target for the Salmonella (Salmonella enterica Typhimurium) effector protein SifA (Salmonella-induced filament protein A) that together regulate the Salmonella-containing vacuole (Fig. 1B). Open in a separate windowFigure 1.Model of PLEKHM1 function in the endocytic and autophagic pathways. (A) Domain structure of PLEKHM1 and their interactions. RUN (RUNDC3A/RPIP8, UNC-14 and RUSC1/NESCA); PH1 and PH2 (Pleckstrin homology domain 1 and 2); C1/Zinc finger (C1); HOPS (homotypic fusion and protein sorting). (B) Proposed positioning of PLEKHM1 and its associated complexes in the autophagy and endocytic pathway. PLEKHM1 localizes to late endosomes and lysosomes in an RAB7-dependent manner. The interaction between PLEKHM1, RAB7, and HOPS on vesicles positions these vesicles for tethering and fusion with autophagosomes, through direct interaction with MAP1LC3/GABARAP proteins. The autophagosomes may also fuse with late endosomes/MVBs (multivesicular bodies) in a PLEKHM1-RAB7-HOPS-dependent manner to produce amphisomes, prior to fusion with the lysosome. PLEKHM1-RAB7-HOPS can also be subverted by the Salmonella effector SifA, for the proper maintenance of the Salmonella-containing vacuole (SCV) and Sif (Salmonella-induced filament) formation. mAtg8s, MAP1LC3/GABARAP proteins.Using a 2-pronged approach, we identified PLEKHM1 as an interaction partner of RAB7 in its GTP-bound active state, RAB7(GTP), and MAP1LC3/GABARAP proteins. PLEKHM1 interacts directly with all MAP1LC3/GABARAP proteins through a highly conserved LC3-interaction motif (LIR) located between the Pleckstrin homology domain 1 (PH1) and PH2 domains of PLEKHM1 (Fig. 1A). Endogenous PLEKHM1 colocalizes with LAMP1 at the cytosolic-facing membrane, but not the lumenal side, of LC3B-containing amphisomes/autolysosomes, indicating that PLEKHM1 is an autophagy adaptor protein rather than a selective cargo receptor.Using SILAC (stable isotope labeling of cells in culture)-labeled inducible PLEKHM1 cells, we identified the HOPS complex as a significant interaction partner. The hexameric HOPS complex is an essential component of the late endocytic fusion machinery and is required for autolysosome formation. PLEKHM1 interacts directly with the HOPS complex, mediated by the RUN domain of PLEKHM1 and the C terminus of VPS39 (Fig. 1A) Crucially, PLEKHM1 forms an endogenous complex with HOPS. In the context of vesicle fusion, the HOPS complex acts as a tether to anchor and position the vesicles prior to fusion that is driven by SNARE proteins. Multiple SNARE proteins, such as VAMP7, VAMP8, VTI1B, SNAP29, and STX17 have been described to be required for autophagosome-lysosome fusion. Upon autophagy induction, enhanced PLEKHM1 coprecipitation is detected with the HOPS complex and the autophagosome specific SNARE STX17, reinforcing a role for PLEKHM1 in autophagosome-lysosome fusion.Both RAB7 and the HOPS complex are integral components of the endocytic pathway and, as such, we wanted to test the effect of PLEKHM1 loss on EGFR (epidermal growth factor receptor) degradation. We used 2 epithelial cell lines, HeLa and Hke3. In both instances, loss of PLEKHM1 causes a marked decrease in the rate of EGFR degradation and increases retention in early endosomes. This is in stark contrast to previous reports that used A549 cells and showed that a lack of PLEKHM1 accelerates EGFR degradation. Clearly, cell lines and their background mutations will have to be considered for future studies.In addition to the endocytic pathway, RAB7 and the HOPS complex are essential for the autophagosome-to-autolysosome transition. Therefore, we also wanted to explore this facet of PLEKHM1 function. We generated Plekhm1 KO MEFs to analyze the effects of autophagy flux in the absence of PLEKHM1. Plekhm1 KO MEFs show a block in autophagy, with the accumulation of SQSTM1/p62 and LC3B-II and, using tandem-fluorescence-LC3B as a marker, a decrease in autolysosome formation. Taken together, these findings suggest that PLEKHM1 functions at the point of autophagosome-lysosome fusion (Fig. 1B).Finally, we were interested in testing the functional role of PLEKHM1, and in particular the LIR, during selective autophagy of protein aggregates. We treated control and PLEKHM1-depleted cells with puromycin and observed aggregate clearance over time after puromycin removal. Cells lacking PLEKHM1 and those reconstituted with a PLEKHM1-LIR mutant were unable to efficiently remove SQSTM1-ubiquitin-positive aggregates, compared to control or PLEKHM1-wild type reconstituted cells, indicating an important role for the final stages of endosome and autophagosome maturation (Fig. 1B).“No man is an island, entire of itself” seems of particular prudence when considering the intertwined nature of both autophagic and endocytic pathways. Indeed, it is interesting that there are multiple RAB7 effector proteins functioning at the late endocytic step that also contribute to autophagy, including FYCO1, KIAA0226/Rubicon, UVRAG and now PLEKHM1, where only PLEKHM1 and UVRAG have been shown to interact with the HOPS complex. All of which, when mutated or depleted, have effects on both the endocytic and autophagic pathways. Clearly the roles of these proteins in cell-type and tissue-specific settings have to be determined before we fully comprehend the complexities of how the endosomal and autophagic pathways integrate and communicate with each other.     

Use of an ion-exchange sponge to immobilise yeast in high gravity apple based (cider) alcoholic fermentations

Summary An ion-exchange sponge that can have a tailored surface charge has been used for yeast immobilisation in high original gravity (o.g. 1.106) cider fermentation. Continuous circulation of fermentation medium through columns containing weakly basic sponge encouraged yeast growth, decreased batch fermentation time and increased final ethanol concentration, possibly aided by sponge enhanced CO₂ removal from solution.     

Very high gravity (VHG) ethanolic brewing and fermentation: a research update

There have been numerous developments in ethanol fermentation technology since the beginning of the new millennium as ethanol has become an immediate viable alternative to fast-depleting crude reserves as well as increasing concerns over environmental pollution. Nowadays, although most research efforts are focused on the conversion of cheap cellulosic substrates to ethanol, methods that are cost-competitive with gasoline production are still lacking. At the same time, the ethanol industry has engaged in implementing potential energy-saving, productivity and efficiency-maximizing technologies in existing production methods to become more viable. Very high gravity (VHG) fermentation is an emerging, versatile one among such technologies offering great savings in process water and energy requirements through fermentation of higher concentrations of sugar substrate and, therefore, increased final ethanol concentration in the medium. The technology also allows increased fermentation efficiency, without major alterations to existing facilities, by efficient utilization of fermentor space and elimination of known losses. This comprehensive research update on VHG technology is presented in two main sections, namely VHG brewing, wherein the effects of nutrients supplementation, yeast pitching rate, flavour compound synthesis and foam stability under increased wort gravities are discussed; and VHG bioethanol fermentation studies. In the latter section, aspects related to the role of osmoprotectants and nutrients in yeast stress reduction, substrates utilized/tested so far, including saccharide (glucose, sucrose, molasses, etc.) and starchy materials (wheat, corn, barley, oats, etc.), and mash viscosity issues in VHG bioethanol production are detailed. Thereafter, topics common to both areas such as process optimization studies, mutants and gene level studies, immobilized yeast applications, temperature effect, reserve carbohydrates profile in yeast, and economic aspects are discussed and future prospects are summarized.     

Conformation of DNA modified at a d(GG) or a d(AG) site by the antitumor drug cis-diamminedichloroplatinum(II)

The purpose of this work was the comparison of the conformational changes induced in the double helix by the adducts formed at d(GG) and d(AG) sites in the reaction between the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP) and DNA. Two duplexes (20-mer) containing either a single d(A*G*) or a single d(G*G) adduct were studied by means of gel electrophoresis and artificial nuclease and chemical probes. It is shown that the d(G*G*) and the d(A*G*) adducts bend DNA similarly, but at the nucleotide level they distort differently the double helix. We suggest that the weaker interactions between platinated A residues and the other nucleotides, as compared to the interactions between platinated G residues and the other nucleotides, are largely responsible for the differences in the distortions induced in DNA by the d(A*G) and d(G*G*) adducts. This suggestion is supported by the study of the distortions induced in duplexes by the d(G*G*) adducts, one of the platinated G residues being paired with a T residue.     

Zinc fertilisation increases tolerance to Rhizoctonia solani (AG 8) in Medicago truncatula

The effect of Zn fertilisation on tolerance of Medicago truncatula to infection by the root-rotting pathogen Rhizoctonia solani (AG 8) was studied in a field survey and in two experiments in controlled conditions. From the field survey, the concentration of Zn in the shoots of medics was found to be inversely related to the severity of disease on the root. Overall, the addition of Zn to Zn-deficient soil in controlled environment experiments resulted in reduced yield loss in the presence of R. solani, a reduction in disease score and no change in the concentration of nutrients in the shoots. However, under Zn deficiency, increasing levels of added R. solani resulted in significant yield loss, an increase in disease score and a reduction in concentration of Zn in the roots. This occurred despite a decrease in the number of infection sites caused by the fungus on the root and a lower amount of R. solani DNA extracted in medics deficient in Zn compared with plants supplied with Zn. While plants supplied with Zn were able to maintain a stable concentration of Zn in the shoots, the concentration of Zn in the roots also declined with increasing levels of R. solani. In conclusion, Zn application does not directly inhibit infection by R. solani, nor reduce its pathogenicity, but it does strongly increase root growth. The net result is that Zn-sufficient plants are more tolerant to the effects of root pruning by the fungus than Zn-deficient plants.     

Production of 21% (v/v) ethanol by fermentation of very high gravity (VHG) wheat mashes

Summary Very high gravity wheat mashes containing 300 g or more sugares per liter were prepared by enzymatic hydrolysis of starch and fermented with a commercial preparation of active dry yeast. The active dry yeast used in this study was a blend of several strains ofSaccharomyces cerevisiae. The fermentation was carried out at 20°C at different pitching rates (inoculation levels) with and without the addition of yeast extract as nutrient supplement. At a pitching rate of 76 million cells per g of mash an ethanol yield of 20.4% (v/v) was obtained. To achieve this yeast extract must be added to the wheat mash as nutrient supplement. When the pitching rate was raised to 750 million cells per g of mash, the ethanol yield increased to 21.5% (v/v) and no nutrient supplement was required. The efficiency of conversion of sugar to ethanol was 97.6% at the highest pitching rate. This declined slightly with decreasing pitching rate. A high proportion of yeast cells lost viability at high pitching rates. It is suggested that nutrients released from yeast cells that lost viability and lysed, contributed to the high yield of ethanol in the absence of any added nutrients.     

EXAFS studies of Fe(III)-phosvitin at high metal to protein ratios

The stereochemistry of the Fe(III) binding sites in chicken egg phosvitin (PST) at very high iron content, in solution and as a powder, has been investigated through EXAFS spectroscopy. We found that the EXAFS spectra obtained for aqueous PST solutions at metal:protein ratios of 20:1 and 40:1 are very similar to those previously obtained by us on a Fe₁₀PST sample. In all cases the iron ions are octahedrally coordinated by oxygen atoms of the serine-bound phosphate groups and by other ligands from either the protein or the solvent. The average metal-donor atom distance is 1.94 Å. At variance, the EXAFS results for a Fe₅₀PST powder sample suggest the occurrence of a switch in iron coordination from octahedral to lower coordination numbers (5,4). The average iron-oxygen distance is virtually unchanged; apparently, four iron ligands are provided by four different coordinate phosphate groups from the phosphorylated serine residues abundant in the protein. This finding contains interesting implications for the structure-function relationships of this intriguing protein.     

Nonenzymatic hydrolysis of adenosinetriphosphate (ATP) at high temperatures and high pressures

Marine organisms living in the deep sea near to hot wells show a fascinating tolerance to extremely high temperatures and pressures. Under the given conditions the synthesis and stability of biomolecules seem to be limiting facts for the basis of life. We studied the influence of high pressures and high temperatures on the hydrolysis of ATP, a universal component for the storage of energy in all known organisms and therefore an extremely interesting and important molecule. The hydrolysis of ATP, ADP and AMP was studied in unbuffered solutions at temperatures between 353 and 369 K at pH values between 3.4 and 10.0. The pressure dependence was determined to p(max) = 220 MPa also at pH 5. All data can be explained by a proton catalyzed mechanism that removes in consecutive steps the final phosphate group. In none of the experiments could pyrophosphate be detected. The influence of phosphate and magnesium ions on the hydrolysis is discussed.     

草鱼(AG)微卫星标记克隆及特征分析

报道了一种简便高效克隆微卫星序列的方法。这一方法的实施步骤包括小片段基因组文库构建、三螺旋捕获、磁珠分离和PCR 扫描。草鱼AG 重复文库的冗余率为7.2%, 富集效率为60.25 倍, PCR 扫描的准确率达100%, 采用这一方法克隆到的AG 重复序列中完美型、非完美型和混合型的比例分别为66.87%、17.17%和15.96%, 不间断重复序列长度大于30 bp 的微卫星座位占51%。34 个微卫星座位中有25 个表现出多态性,PIC 值0.50—0.91, 进一步分析表明AG 重复序列的多态信息含量(PIC)与不间断重复序列长度相关, 不间断重复序列长度大于30 bp 的座位表现出丰富的多态性。较高的富集率和有效引物获得率证明此方法在分离草鱼微卫星中的成功应用, 并将为草鱼养殖品系的优化、遗传多样性的检测及遗传图谱的构建等打下基础。       

Angiogenic activity of syndecan-binding laminin peptide AG73 (RKRLQVQLSIRT)

The AG73 peptide (RKRLQVQLSIRT, mouse laminin alpha 1 chain 2719-2730) promotes cell adhesion and tumor metastasis, and interacts with transmembrane syndecan proteoglycans. Here, we demonstrate AG73 peptide angiogenic activity using in vitro, ex vivo, and in vivo models. AG73 induced murine endothelial cell (SVEC4-10) tube formation on Cultrex Basement Membrane Extract (Cultrex BME) and stimulated sprouting of aortic rings. None of the homologous sequences from the laminin alpha2, alpha3, alpha4, or alpha5 chains was as active as AG73 in promoting sprouting formation. AG73 also mediated angiogenesis in the chick chorioallantonic membrane (CAM) assay. Using subcutaneously injected Cultrex BME supplemented with AG73, we observed a large angiogenic response. Furthermore, AG73-conjugated to a chitosan membrane promoted a strong angiogenic response in the CAM assay. These results indicate that the AG73 peptide is a potent syndecan-binding angiogenesis stimulator and may be useful for therapeutic application to treat ischemic injuries.     

Deionization of formamide with Biorad AG501-X(D)

We have studied different conditions of the deionization of formamide with Biorad mixed bed resin AG501-X(D) and find that contrary to the popular usage, drying the resin before deionization produces the best results. In a typical deionization procedure, conductivity initially goes down, reaches a minimum plateau and finally goes up again. The initial rate of deionization, the minimum conductivity reached at the plateau and the rate of final rise in conductivity depend on whether the resin is dry or wet (i.e., straight from the bottle) and on the amount of resin used. In general, wet resin produced faster initial deionization, higher minimum conductivity and quicker final rise in conductivity. Surprisingly, a smaller amount (5%) of resin worked better than a larger amount (20%). With smaller amount of resin, although initial deionization was slower, the minimum conductivity achieved was lower and the final rise in conductivity was slower. This was partly due to the fact that the conductivity of formamide increases faster with increasing amount of water in it.     

Improvement of the ethanol productivity in a high gravity brewing at pilot plant scale

A 2³ full factorial design was used to study the influence of different experimental variables, namely wort gravity, fermentation temperature and nutrient supplementation, on ethanol productivity from high gravity wort fermentation by Saccharomyces cerevisiae (lager strain), under pilot plant conditions. The highest ethanol productivity (0.69 g l^–1 h^–1) was obtained at 20°P [°P is the weight of extract (sugar) equivalent to the weight of sucrose in a 100 g solution at 20°C], 15°C, with the addition of 0.8% (w/v) yeast extract, 24 mg l^–1 ergosterol and 0.24% (v/v) Tween 80.