Carbon balance in leaves of young poplar trees

In the present study, important components of carbon metabolism of mature leaves of young poplar trees (Populus x canescens) were determined. Carbohydrate concentrations in leaves and xylem sap were quantified at five different times during the day and compared with photosynthetic gas exchange measurements (net assimilation, transpiration and rates of isoprene emission). Continuously measured xylem sap flow rates, with a time resolution of 15 min, were used to calculate diurnal balances of carbon metabolism of whole mature poplar leaves on different days. Loss of photosynthetically fixed carbon by isoprene emission and dark respiration amounted to 1% and 20%. The most abundant soluble carbohydrates in leaves and xylem sap were glucose, fructose and sucrose, with amounts of approx. 2 to 12 mmol m(-2) leaf area in leaves and about 0.2 to 15 mM in xylem sap. Clear diurnal patterns of carbohydrate concentration in xylem sap and leaves, however, were not observed. Calculations of the carbon transport rates in the xylem to the leaves were based on carbohydrate concentrations in xylem sap and xylem sap flow rates. This carbon delivery amounted to about 3 micromol C m(-2) s(-1) during the day and approx. 1 micromol C m(-2) s(-1) at night. The data demonstrated that between 9 and 28 % of total carbon delivered to poplar leaves during 24 h resulted from xylem transport and, hence, provide a strong indication for a significant rate of carbon cycling within young trees.     

Osmoregulated periplasmic glucans of Erwinia chrysanthemi

We report the initial characterization of the osmoregulated periplasmic glucans (OPGs) of Erwinia chrysanthemi. OPGs are intrinsic components of the bacterial envelope necessary to the pathogenicity of this phytopathogenic enterobacterium (F. Page, S. Altabe, N. Hugouvieux-Cotte-Pattat, J.-M. Lacroix, J. Robert-Baudouy and J.-P. Bohin, J. Bacteriol. 183:0000-0000, 2001 [companion in this issue]). OPGs were isolated by trichloracetic acid treatment and gel permeation chromatography. The synthesis of these compounds appeared to be osmoregulated, since lower amounts of OPGs were produced when bacteria were grown in media of higher osmolarities. However, a large fraction of these OPGs were recovered in the culture medium. Then, these compounds were characterized by compositional analysis, high-performance anion-exchange chromatography, matrix-assisted laser desorption mass spectrometry, and (1)H and (13)C nuclear magnetic resonance analyses. OPGs produced by E. chrysanthemi are very heterogeneous at the level of both backbone structure and substitution of these structures. The degree of polymerization of the glucose units ranges from 5 to 12. The structures are branched, with a linear backbone consisting of beta-1,2-linked glucose units to which a variable number of branches, composed of one glucose residue, are attached by beta-1,6 linkages in a random way. This glucan backbone may be substituted by O-acetyl and O-succinyl ester-linked residues.     

Osmoregulated periplasmic glucan synthesis is required for Erwinia chrysanthemi pathogenicity

Erwinia chrysanthemi is a phytopathogenic enterobacterium causing soft rot disease in a wide range of plants. Osmoregulated periplasmic glucans (OPGs) are intrinsic components of the gram-negative bacterial envelope. We cloned the opgGH operon of E. chrysanthemi, encoding proteins involved in the glucose backbone synthesis of OPGs, by complementation of the homologous locus mdoGH of Escherichia coli. OpgG and OpgH show a high level of similarity with MdoG and MdoH, respectively, and mutations in the opgG or opgH gene abolish OPG synthesis. The opg mutants exhibit a pleiotropic phenotype, including overproduction of exopolysaccharides, reduced motility, bile salt hypersensitivity, reduced protease, cellulase, and pectate lyase production, and complete loss of virulence. Coinoculation experiments support the conclusion that OPGs present in the periplasmic space of the bacteria are necessary for growth in the plant host.     

Degree of sex reversal as related to plasma steroid levels in genetic female chickens (Gallus domesticus) treated with Fadrozole

The objectives of this work were to determine whether or not plasma levels of testosterone and estradiol reflect the various grades of sex reversal in genetic female chickens treated with Fadrozole (CGS 16949 A), a nonsteroidal aromatase inhibitor, and whether gonadal aromatase activity and plasma levels of testosterone and estradiol in treated females can or not be modified by post-hatch treatments with Fadrozole or Fadrozole + testosterone. Eggs were injected with 1 mg Fadrozole on day 4 of incubation. In females having developed sex-reversed gonads, endocrine parameters (estradiol and testosterone) at and after 13 weeks of age were indicative of the degree of sex reversal, with, for example, sex-reversed females with two testes having the highest levels of testosterone and the lowest levels of estradiol. Among these females, eight (from a total of 13) produced ejaculates with scarce and abnormal spermatozoa. Some motility was observable in the ejaculates from five of them. None of the post-hatch treatments had a significant effect on plasma levels of testosterone or estradiol (measured at 3-week intervals from week 4 to week 28 post-hatch) or on gonadal aromatase activity (measured at 12 and 28 weeks). In conclusion, these results indicate that plasma levels of testosterone and estradiol at and after 13 weeks of age are valuable indicators of the degree of sex reversal in female chickens treated with Fadrozole prior to gonadal sex differentiation. In pre-cited conditions, post-natal treatments with either Fadrozole or Fadrozole + testosterone had no apparent effect on the degree of sex reversal in these birds. Finally, the occurrence of ejaculates with motile although scarce and abnormal spermatozoa, revealed that epididymes and ducti deferens can develop and become functional in sex-reversed female chickens.     

The importance of the Q motif in the ATPase activity of a viral helicase

NS3 proteins of flaviviruses contain motifs which indicate that they possess protease and helicase activities. The helicases are members of the DExD/H box helicase superfamily and NS3 proteins from some flaviviruses have been shown to possess ATPase and helicase activities in vitro. The Q motif is a recently recognised cluster of nine amino acids common to most DExD/H box helicases which is proposed to regulate ATP binding and hydrolysis. In addition a conserved residue occurs 17 amino acids upstream of the Q motif ('+17'). We have analysed full-length and truncated NS3 proteins from Powassan virus (a tick-borne flavivirus) to investigate the role that the Q motif plays in the hydrolysis of ATP by a viral helicase. The Q motif appears to be essential for the activity of Powassan virus NS3 ATPase, however NS3 deletion mutants that contain the Q motif but lack the '+17' amino acid have ATPase activity albeit at a reduced level.     

Ectopic pacing at physiological rate improves postanoxic recovery of the developing heart

Recently, rapid and transient cardiac pacing was shown to induce preconditioning in animal models. Whether the electrical stimulation per se or the concomitant myocardial ischemia affords such a protection remains unknown. We tested the hypothesis that chronic pacing of a cardiac preparation maintained in a normoxic condition can induce protection. Hearts of 4-day-old chick embryos were electrically paced in ovo over a 12-h period using asynchronous and intermittent ventricular stimulation (5 min on-10 min off) at 110% of the intrinsic rate. Sham (n = 6) and paced hearts (n = 6) were then excised, mounted in vitro, and subjected successively to 30 min of normoxia (20% O(2)), 30 min of anoxia (0% O(2)), and 60 min of reoxygenation (20% O(2)). Electrocardiogram and atrial and ventricular contractions were simultaneously recorded throughout the experiment. Reoxygenation-induced chrono-, dromo-, and inotropic disturbances, incidence of arrhythmias, and changes in electromechanical delay (EMD) in atria and ventricle were systematically investigated in sham and paced hearts. Under normoxia, the isolated heart beat spontaneously and regularly, and all baseline functional parameters were similar in sham and paced groups (means +/- SD): heart rate (190 +/- 36 beats/min), P-R interval (104 +/- 25 ms), mechanical atrioventricular propagation (20 +/- 4 mm/s), ventricular shortening velocity (1.7 +/- 1 mm/s), atrial EMD (17 +/- 4 ms), and ventricular EMD (16 +/- 2 ms). Under anoxia, cardiac function progressively collapsed, and sinoatrial activity finally stopped after approximately 9 min in both groups. During reoxygenation, paced hearts showed 1) a lower incidence of arrhythmias than sham hearts, 2) an increased rate of recovery of ventricular contractility compared with sham hearts, and 3) a faster return of ventricular EMD to basal value than sham hearts. However, recovery of heart rate, atrioventricular conduction, and atrial EMD was not improved by pacing. Activity of all hearts was fully restored at the end of reoxygenation. These findings suggest that chronic electrical stimulation of the ventricle at a near-physiological rate selectively alters some cellular functions within the heart and constitutes a nonischemic means to increase myocardial tolerance to a subsequent hypoxia-reoxygenation.     

Seasonal gonad progesterone pattern in the soft-shell clam Mya arenaria

Steroidogenesis, which plays a major role in the reproductive cycle of vertebrates, is still for the most part, unknown in invertebrates. The aim of this study was to examine the link between progesterone and the reproductive cycle in Mya arenaria. The soft-shell clams, Mya arenaria were collected in Anse à l'Orignal (Parc Provincial du Bic, Québec, Canada) from July to November 1998. Histological data have shown that female gonads of M. arenaria were in the spawning stage in August and September, while the male gonads were in the ripe stage. This period of active gametogenesis was associated with a depletion of lipid reserves. These lipids could be used as a source of energy and as a substrate for steroidogenesis. Liquid Chromatography coupled to Mass Spectroscopy (LC-MS) and quantified by Enzyme Linked ImmunoSorbent Assay (ELISA) determined progesterone. Progesterone levels in the gonad were increased during the ripe stage in the male and during the spawning stage in the female. These results indicate, for the first time, that progesterone, as in vertebrates, may play a role in the reproductive cycle of M. arenaria.     

Inhibitors of protein-disulfide isomerase prevent cleavage of disulfide bonds in receptor-bound glycoprotein 120 and prevent HIV-1 entry

We previously reported that monoclonal antibodies to protein-disulfide isomerase (PDI) and other membrane-impermeant PDI inhibitors prevented HIV-1 infection. PDI is present at the surface of HIV-1 target cells and reduces disulfide bonds in a model peptide attached to the cell membrane. Here we show that soluble PDI cleaves disulfide bonds in recombinant envelope glycoprotein gp120 and that gp120 bound to the surface receptor CD4 undergoes a disulfide reduction that is prevented by PDI inhibitors. Concentrations of inhibitors that prevent this reduction and inhibit the cleavage of surface-bound disulfide conjugate prevent infection at the level of HIV-1 entry. The entry of HIV-1 strains differing in their coreceptor specificities is similarly inhibited, and so is the reduction of gp120 bound to CD4 of coreceptor-negative cells. PDI inhibitors also prevent HIV envelope-mediated cell-cell fusion but have no effect on the entry of HIV-1 pseudo-typed with murine leukemia virus envelope. Importantly, PDI coprecipitates with both soluble and cellular CD4. We propose that a PDI.CD4 association at the cell surface enables PDI to reach CD4-bound virus and to reduce disulfide bonds present in the domain of gp120 that binds to CD4. Conformational changes resulting from the opening of gp120-disulfide loops may drive the processes of virus-cell and cell-cell fusion. The biochemical events described identify new potential targets for anti-HIV agents.     

Plant mitochondria move on F-actin, but their positioning in the cortical cytoplasm depends on both F-actin and microtubules

Mitochondrion movement and positioning was studied in elongating cultured cells of tobacco (Nicotiana tabacum L.), containing mitochondria-localized green fluorescent protein. In these cells mitochondria are either actively moving in strands of cytoplasm transversing or bordering the vacuole, or immobile positioned in the cortical layer of cytoplasm. Depletion of the cell's ATP stock with the uncoupling agent DNP shows that the movement is much more energy demanding than the positioning. The active movement is F-actin based. It is inhibited by the actin filament disrupting drug latrunculin B, the myosin ATPase inhibitor 2,3-butanedione 2-monoxime and the sulphydryl-modifying agent N-ethylmaleimide. The microtubule disrupting drug oryzalin did not affect the movement of mitochondria itself, but it slightly stimulated the recruitment of cytoplasmic strands, along which mitochondria travel. The immobile mitochondria are often positioned along parallel lines, transverse or oblique to the cell axis, in the cortical cytoplasm of elongated cells. This positioning is mainly microtubule based. After complete disruption of the F-actin, the mitochondria parked themselves into conspicuous parallel arrays transverse or oblique to the cell axis or clustered around chloroplasts and around patches and strands of endoplasmic reticulum. Oryzalin inhibited all positioning of the mitochondria in parallel arrays.