Functional RelBE-Family Toxin-Antitoxin Pairs Affect Biofilm Maturation and Intestine Colonization in Vibrio cholerae

Toxin–antitoxin (TA) systems are small genetic elements that typically encode a stable toxin and its labile antitoxin. These cognate pairs are abundant in prokaryotes and have been shown to regulate various cellular functions. Vibrio cholerae, a human pathogen that is the causative agent of cholera, harbors at least thirteen TA loci. While functional HigBA, ParDE have been shown to stabilize plasmids and Phd/Doc to mediate cell death in V. cholerae, the function of seven RelBE-family TA systems is not understood. In this study we investigated the function of the RelBE TA systems in V. cholerae physiology and found that six of the seven relBE loci encoded functional toxins in E. coli. Deletion analyses of each relBE locus indicate that RelBE systems are involved in biofilm formation and reactive oxygen species (ROS) resistance. Interestingly, all seven relBE loci are induced under the standard virulence induction conditions and two of the relBE mutants displayed a colonization defect, which was not due to an effect on virulence gene expression. Although further studies are needed to characterize the mechanism of action, our study reveals that RelBE systems are important for V. cholerae physiology.     

Multiple mechanisms contribute to myenteric plexus ablation induced by benzalkonium chloride in the guinea-pig ileum

Ablation of rat myenteric plexus with benzalkonium chloride has provided a model of intestinal aganglionosis, but the degenerative responses are not well understood. We examined the effects of this detergent on neurons and glia, including expression of c-Myc, c-Jun, JunB, and c-Fos, and on immunocytes in the guinea-pig ileum. Benzalkonium chloride (0.1%) or saline was applied to the serosal surface of distal ileum. Tissues were analyzed 2, 3, or 7 days later and compared with cyclosporine-treated and untreated animals. More than 90% of myenteric neurons were destroyed in ileal segments 3–7 days after benzalkonium-chloride treatment. Glia withdrew processes from around neurons after 2 days and were mostly gone after 3 days. Neuronal c-Myc began to disappear while c-Fos, c-Jun, and JunB were evident in some neuronal nuclei after 2 or 3 days. After 3 days, widespread apoptosis was evident in the myenteric plexus. Populations of T cells, B cells, and macrophage-like cells in untreated and saline-treated myenteric plexuses were substantially increased 3 and 7 days after benzalkonium-chloride treatment. Cyclosporine delayed significant neuronal loss. We conclude that a variety of degenerative mechanisms may be active in this model, including an immune response which may actively contribute to tissue destruction. Received: 13 September 1996 / Accepted: 20 January 1997     

Effect of redox-responsive DTSSP crosslinking on poly(l-lysine)-grafted-poly(ethylene glycol) nanoparticles for delivery of proteins

Therapeutic proteins are utilized in a variety of clinical applications, but side effects and rapid in vivo clearance still present hurdles. An approach that addresses both drawbacks is protein encapsulation within in a polymeric nanoparticle, which is effective but introduces the additional challenge of destabilizing the nanoparticle shell in clinically relevant locations. This study examined the effects of crosslinking self-assembled poly(l -lysine)-grafted-poly(ethylene glycol) nanoparticles with redox-responsive 3,3′-dithiobis(sulfosuccinimidyl propionate) (DTSSP) to achieve nanoparticle destabilization in a reductive environment. The polymer-protein nanoparticles (DTSSP NPs) were formed through electrostatic self-assembly and crosslinked with DTSSP, which contains a glutathione-reducible disulfide. As glutathione is upregulated in various cancers, DTSSP NPs could display destabilization within cancer cells. A library of DTSSP NPs was formed with varying copolymer to protein (C:P) and crosslinker to protein (X:P) mass ratios and characterized by size and encapsulation efficiency. DTSSP NPs with a 7:1 C:P ratio and 2:1 X:P ratio were further characterized by stability in the presence proteases and reducing agents. DTSSP NPs fully encapsulated the model protein and displayed 81% protein release when incubated with 5 mM dithiothreitol for 12 hr. This study contributes to understanding stimulus-responsive crosslinking of polymeric nanoparticles and could be foundational to clinical administration of therapeutic proteins.     

Low oxygen inhibition of photosynthesis is caused by inhibition of starch synthesis

Photosynthesis of C₃ plants is occasionally inhibited upon switching from normal to low partial pressure of O₂. Leaves of Solanum tuberosum exhibited this effect reproducibly under saturating light and 700 microbars of CO₂. We determined the partitioning of recent photosynthate between starch and sucrose and measured the concentration of hexose monophosphates in the stroma and cytosol after nonaqueous fractionation. The reduction in the rate of photosynthesis upon switching to low partial pressure of O₂ was caused by reduced starch synthesis. The concentration of hexose monophosphates in the stroma fell and the glucose 6-phosphate to fructose 6-phosphate to fructose 6-phosphate ratio fell from 2.7 to 1.3, indicating an inhibition of phosphoglucoisomerase as described by K-J Dietz ([1985] Biochim Biophys Acta 839: 240-248). The concentration of hexose monophosphates in the cytosol increased, ruling out a sucrose synthesis limitation by reduced transport from the chloroplast as the explanation for low O₂ inhibition of photosynthesis.     

Estimating the rate of photorespiration in leaves

The influence of Li⁺ on the circumnutations of hypocotyls of Helianthus annuus L . cv. Californicus was investigated. LiCl at concentration levels from 0 to 40 m M (lethal) was added to intact hypocotyls grown in liquid nutrient medium. The Li⁺ concentration in the hypocotyls was measured by flame photometry. The growth of the hypocotyls was not affected by the LiCl.
Amplitude and frequency of the circumnutations were determined by correlation analysis. The oscillatory pattern of the movements became less regular at concentrations above 10 m M LiCl. The amplitude of the movements was reduced for concentrations above 7 m M LiCl. The frequency of the movements was reduced when LiCl was increased from 0 to 10 m M . Above 10 m M LiCl the frequency of the circumnutations was higher than for control plants. The results showed that circumnutations of sunflower hypocotyls can be added to the group of oscillators in biological organisms that are affected by Li⁺.     

Reduced Cytosolic Fructose-1,6-Bisphosphatase Activity Leads to Loss of O(2) Sensitivity in a Flaveria linearis Mutant

The mutant plant of Flaveria linearis characterized by Brown et al. (Plant Physiol. 81: 212-215) was studied to determine the cause of the reduced sensitivity to O₂. Analysis of CO₂ assimilation metabolites of freeze clamped leaves revealed that both 3-phosphoglycerate and ribulose 1,5-bisphosphate were high in the mutant plant relative to F. linearis with normal O₂ sensitivity. The k_cat of ribulose-1,5-bisphosphate carboxylase (RuBPCase) was equal in all plant material tested (range 18-22 s⁻¹) indicating that no tight binding inhibitor was present. The degree of RuBPCase carbamylation was reduced in the mutant plant relative to the wild-type plant. Since 3-phosphoglycerate was high in the mutant plant and photosynthesis did not exhibit properties associated with RuBPCase limitations, we believe that the decarbamylation of RuBPCase was a consequence of another lesion in photosynthesis. Fructose 1,6-bisphosphate and its precursors, such as the triose phosphates, were in high concentration in the mutant plant relative to the wild type. The concentrations of the product of the fructose 1,6-bisphosphatase reaction, fructose 6-phosphate, and its isomer, glucose 6-phosphate, were the same in both plants. We found that the mutant plant had up to 75% less cytosolic fructose 1,6-bisphosphatase activity than the wild type but comparable levels of stromal fructose 1,6-bisphosphatase. We conclude that the reduced fructose-1,6-bisphosphatase activity restricts the mutant plant's capacity for sucrose synthesis and this leads to reduced or reversed O₂ sensitivity.     

The in-vivo response of the ribulose-1,5-bisphosphate carboxylase activation state and the pool sizes of photosynthetic metabolites to elevated CO2 inPhaseolus vulgaris L.

The short-term, in-vivo response to elevated CO₂ of ribulose-1,5-bisphosphate carboxylase (RuBPCase, EC 4.1.1.39) activity, and the pool sizes of ribulose 1,5-bisphosphate, 3-phosphoglyceric acid, triose phosphates, fructose 1,6-bisphosphate, glucose 6-phosphate and fructose 6-phosphate in bean were studied. Increasing CO₂ from an ambient partial pressure of 360–1600 bar induced a substantial deactivation of RuBPCase at both saturating and subsaturating photon flux densities. Activation of RuBPCase declined for 30 min following the CO₂ increase. However, the rate of photosynthesis re-equilibrated within 6 min of the switch to high CO₂, indicating that RuBPCase activity did not limit photosynthesis at high CO₂. Following a return to low CO₂, RuBPCase activation increased to control levels within 10 min. The photosynthetic rate fell immediately after the return to low CO₂, and then increased in parallel with the increase in RuBPCase activation to the initial rate observed prior to the CO₂ increase. This indicated that RuBPCase activity limited photosynthesis while RuBPCase activation increased. Metabolite pools were temporarily affected during the first 10 min after either a CO₂ increase or decrease. However, they returned to their original level as the change in the activation state of RuBPCase neared completion. This result indicates that one role for changes in the activation state of RuBPCase is to regulate the pool sizes of photosynthetic intermediates.Abbreviations and symbols A net CO₂ assimilation rate - C_a ambient CO₂ partial pressure - C_i intercellular CO₂ partial pressure - CABP 2-carboxyarabinitol 1,5-bisphosphate - k_cat catalytic turnover rate per RuBPCase molecule - PFD photon flux density (400 to 700 nm on an area basis) - PGA 3-phosphoglyceric acid - P_i orthophosphate - RuBP ribulose 1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39)     

PHYLOGENY OF THE SUBFAMILIES OF THE FAMILY BRACONIDAE (HYMENOPTERA: ICHNEUMONOIDEA): A REASSESSMENT

Abstract— The recently published phylogeny of Braconidae by Quicke and van Achterberg is reassessed. Character-state definitions and character polarities are evaluated, and more rigorous methods are suggested. Our results indicate that there are many more parsimonious solutions to their data set, the consensus of which differs substantially from their results. Based on our reassessment, little can be said about the relationships among braconid subfamilies. Consensus trees show the cyclostomes as a largely unresolved basal grade. The two other major lineages which have been proposed, the helconoids and microgastroids, are somewhat better resolved, but not consistently so. Relationships among the helconoids vary considerably depending on the parameters used for parsimony analysis.