Role of the histidine kinase, EnvZ, in the production of outer membrane proteins in the symbiotic-pathogenic bacterium Xenorhabdus nematophilus.

We show that inactivation of envZ, the gene encoding the histidine kinase sensor protein, EnvZ, of Xenorhabdus nematophilus, affected the production of several outer membrane proteins (Opns). X. nematophilus produced five major Opns during exponential growth. Insertional inactivation of envZ led to a decrease in the production of OpnP, the OmpF-like pore-forming protein which constitutes approximately 50% of the total outer membrane protein in X. nematophilus. OpnA production was also reduced, while the remaining Opns were produced normally. During the transition to stationary phase, three new outer membrane proteins, OpnB, OpnS, and OpnX, were induced in the wild-type strain. The envZ-minus strain, ANT1, did not produce OpnB and OpnX, while OpnS was induced at markedly reduced levels. These results suggest that EnvZ was required for the high-level production of OpnP during exponential growth and may be involved in the production of OpnB, OpnS, and OpnX during stationary-phase growth. We also show that ANT1 was more pathogenic than the wild-type strain when as few as five cells were injected into the hemolymph of the larval stage of the tobacco hornworm (Manduca sexta). The larvae died before significant numbers of bacteria were detectable in the hemolymph. These results are discussed in relation to the role of EnvZ in the life cycle of X. nematophilus.     

Long-Term Shedding of Influenza Virus,Parainfluenza Virus,Respiratory Syncytial Virus and Nosocomial Epidemiology in Patients with Hematological Disorders

Respiratory viruses are a cause of upper respiratory tract infections (URTI), but can be associated with severe lower respiratory tract infections (LRTI) in immunocompromised patients. The objective of this study was to investigate the genetic variability of influenza virus, parainfluenza virus and respiratory syncytial virus (RSV) and the duration of viral shedding in hematological patients. Nasopharyngeal swabs from hematological patients were screened for influenza, parainfluenza and RSV on admission as well as on development of respiratory symptoms. Consecutive swabs were collected until viral clearance. Out of 672 tested patients, a total of 111 patients (17%) were infected with one of the investigated viral agents: 40 with influenza, 13 with parainfluenza and 64 with RSV; six patients had influenza/RSV or parainfluenza/RSV co-infections. The majority of infected patients (n = 75/111) underwent stem cell transplantation (42 autologous, 48 allogeneic, 15 autologous and allogeneic). LRTI was observed in 48 patients, of whom 15 patients developed severe LRTI, and 13 patients with respiratory tract infection died. Phylogenetic analysis revealed a variety of influenza A(H1N1)pdm09, A(H3N2), influenza B, parainfluenza 3 and RSV A, B viruses. RSV A was detected in 54 patients, RSV B in ten patients. The newly emerging RSV A genotype ON1 predominated in the study cohort and was found in 48 (75%) of 64 RSV-infected patients. Furthermore, two distinct clusters were detected for RSV A genotype ON1, identical RSV G gene sequences in these patients are consistent with nosocomial transmission. Long-term viral shedding for more than 30 days was significantly associated with prior allogeneic transplantation (p = 0.01) and was most pronounced in patients with RSV infection (n = 16) with a median duration of viral shedding for 80 days (range 35–334 days). Long-term shedding of respiratory viruses might be a catalyzer of nosocomial transmission and must be considered for efficient infection control in immunocompromised patients.     

Amidase and urease activities in plants

Summary Foliar fertilization has received considerable attention in recent years. Because of the importance of amides and urea as N sources, this work was carried out to study the enzymes that catalyze the hydrolysis of these compounds in plant leaves. The methods developed for assay of these enzymes in plants involve determination by steam distillation of the NH₄ ⁺–N produced by amidase or urease activity when plant materials are incubated at 37°C with buffered (0.1M THAM pH 8.0) amide solution or buffered (0.1M THAM pH 7.5) urea solution, respectively. Amidase and urease were detected in 21 diverse plants in the families of Gramineae and Leguminosae. Results showed that amidase and urease have optimum activities at buffer pH values of 8.0 and 7.5, respectively. Both amidase and urease activities were decreased significantly upon freezing or air-drying of plant samples before enzyme assay. These differences were proportional to the original activities of fresh plant materials. Studies on the effect of temperature on amidase and urease activities showed that these enzymes are inactivated at temperatures above 60 and 70°C, respectively. The energy of activation of the reaction catalyzed by amidase and urease in plants, expressed in kJ·mole^–1, ranged from 44.0 to 51.2 (avg.=47.1) and from 43.1 to 56.5 (avg.=51.2) when formamide and urea were used as substrates, respectively. The apparent K_m constants of these enzymes varied among the plant samples studied. By using the Lineweaver-Burk plot, the K_m values for amidase when formamide was used as a substrate ranged from 2.0 to 9.4 (avg.=5.8 mM) and for urease ranged from 0.4 to 1.6 (avg.=0.8 mM). The V_max values of 7 plant samples, expressed in g of NH₄ ⁺–N produced/0.1 g of plant materials/2h, ranged from 137 to 514 for amidase and from 29 to 123 for urease. The importance of these enzymes in application of amides and urea to plant leaves is discussed.     

Effects of bupivacaine on the membrane properties of nerve cell soma

While the effects of local anaesthetics on axonal conduction and axonal membrane have been extensively studied, there is little information about the actions of these agents on nerve cell soma. Therefore, the effects of the amide local anaesthetic bupivacaine on the electrophysiologic properties of the nerve cell soma were studied on isolated superfused superior cervical ganglia of rats. Administration of 100-200 nM of bupivacaine to the preparation produced marked changes in membrane properties of the cell soma. The resting membrane potential did not change, but the membrane resistance decreased 20% (P less than 0.01). The firing threshold, the action potential duration at 50% of maximal amplitude, and the intracellular current threshold for firing the cells increased significantly (P less than 0.01), while the action potential amplitude decreased significantly (P less than 0.01), before its complete blockade. The results show that the cell soma is a major site of action of local anaesthetics. The implication of the results is that when local anaesthetics are applied to areas where cell bodies and processes (axons and dendrites) are present together, such as during celiac plexus block, lumbar sympathetic block, stellate ganglion block, etc., they will all be effectively depressed and/or blocked.     

TAXONOMIC DISTRIBUTION OF COPPER-ZINC SUPEROXIDE DISMUTASE IN GREEN ALGAE AND ITS PHYLOGENETIC IMPORTANCE1

Twenty-nine species of green algae (from four classes: Charophyceae, Chlorophyceae, Pleurastrophyceae, and Ulvophyceae, but not the Micromonadophyceae), five species of bryophytes, and four species of vascular plants were tested for the presence of iron, manganese, and copper-zinc superoxide dismutase. Copper-zinc superoxide dismutase was found in members of the Charophyceae, Bryophyta, and Tracheophyta. Manganese and/or iron superoxide dismutase were found in all classes of green algae and most land plants. The presence of the copper-zinc form of superoxide dismutase only in the class of green algae hypothesized to be closest to the land plant progenitors strengthens the argument for that phylogenetic relationship. It also suggests that this form of superoxide dismutase was a vital attribute for the survival of the earliest land plants.     

<Emphasis Type="Italic">Fremyella diplosiphon</Emphasis> as a Biodiesel Agent: Identification of Fatty Acid Methyl Esters via Microwave-Assisted Direct In Situ Transesterification

Increasing concerns on environmental and economic issues linked to fossil fuel use has driven great interest in cyanobacteria as third-generation biofuel agents. In this study, the biodiesel potential of a model photosynthetic cyanobacterium, Fremyella diplosiphon, was identified by fatty acid methyl esters (FAME) via direct transesterification. Total lipids in wild type (Fd33) and halotolerant (HSF33-1 and HSF33-2) strains determined by gravimetric analysis yielded 19% cellular dry weight (CDW) for HSF33-1 and 20% CDW for HSF33-2, which were comparable to Fd33 (18% CDW). Gas chromatography-mass spectrometry detected a high ratio of saturated to unsaturated FAMEs (2.48–2.61) in transesterified lipids, with methyl palmitate being the most abundant (C16:0). While theoretical biodiesel properties revealed high cetane number and oxidative stability, high cloud and pour point values indicated that fuel blending could be a viable approach. Significantly high FAME abundance in total transesterified lipids of HSF33-1 (40.2%) and HSF33-2 (69.9%) relative to Fd33 (25.4%) was identified using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry, indicating that robust salt stress response corresponds to higher levels of extractable FAME. Alkanes, a key component in conventional fuels, were present in F. diplosiphon transesterified lipids across all strains confirming that natural synthesis of these hydrocarbons is not inhibited during biodiesel production. While analysis of photosynthetic pigments and phycobiliproteins did not reveal significant differences, FAME abundance varied significantly in wild type and halotolerant strains indicating that photosynthetic pathways are not the sole factors that determine fatty acid production. We characterize the potential of F. diplosiphon for biofuel production with FAME yields in halotolerant strains higher than the wild type with no loss in photosynthetic pigmentation.     

Transient Inhibition of FGFR2b-Ligands Signaling Leads to Irreversible Loss of Cellular β-Catenin Organization and Signaling in AER during Mouse Limb Development

The vertebrate limbs develop through coordinated series of inductive, growth and patterning events. Fibroblast Growth Factor receptor 2b (FGFR2b) signaling controls the induction of the Apical Ectodermal Ridge (AER) but its putative roles in limb outgrowth and patterning, as well as in AER morphology and cell behavior have remained unclear. We have investigated these roles through graded and reversible expression of soluble dominant-negative FGFR2b molecules at various times during mouse limb development, using a doxycycline/transactivator/tet(O)-responsive system. Transient attenuation (≤24 hours) of FGFR2b-ligands signaling at E8.5, prior to limb bud induction, leads mostly to the loss or truncation of proximal skeletal elements with less severe impact on distal elements. Attenuation from E9.5 onwards, however, has an irreversible effect on the stability of the AER, resulting in a progressive loss of distal limb skeletal elements. The primary consequences of FGFR2b-ligands attenuation is a transient loss of cell adhesion and down-regulation of P63, β1-integrin and E-cadherin, and a permanent loss of cellular β-catenin organization and WNT signaling within the AER. Combined, these effects lead to the progressive transformation of the AER cells from pluristratified to squamous epithelial-like cells within 24 hours of doxycycline administration. These findings show that FGFR2b-ligands signaling has critical stage-specific roles in maintaining the AER during limb development.