Interaction of Colicins with Bacterial Cells III. Colicin-tolerant Mutations in Escherichia coli

Mutants that adsorb certain colicins without being killed, i.e., tolerant mutants (tol), were isolated from Escherichia coli K-12 strains. Selection was done either with colicin K or E2. Several groups of mutants showing different phenotypes were found, and some of them showed tolerance to both K and E colicins, which have different receptors. Many of these mutants mapped near gal. Typical mutants from group II, III, and IV were studied in more detail. The mutant loci were contransducible with gal by phage P1. The linkage order was deduced to be tol-gal-λ. In partially diploid strains, these mutant loci are recessive to wild-type alleles. Temperature-dependent conditionally tolerant mutants were also isolated. Two groups were found: the first was tolerant to E2 and E3 at 40 C, but sensitive at 30 C; the second was tolerant to E2 at 30 C, but sensitive at 40 C. Experiments done with these mutants suggest that these mutations affect the heat lability of some protein that is necessary for the response of cells to colicins. Conditionally lethal tolerant mutants were isolated which at 40 C were tolerant to E2 and E3 and could not grow, but which at 30 C were fully sensitive and grew normally. The mutation mapped near malA. The tolerance at 40 C is not due to a consequence of an inactivation of general cellular metabolism, but presumably is a cause of the subsequent inhibition of cellular growth. The results suggest that some protein components involved in the response to colicin are also vital to normal cellular growth.     

Changes in lung mechanics and reactivity with age after viral bronchiolitis in beagle puppies

We measured changes with growth in lung function and airway reactivity after acute canine parainfluenza virus type 2 (CPI2, n = 5), canine adenovirus type 2 (CAV2, n = 7), and sequential CAV2-CPI2 (n = 6) infections or no infection (controls, n = 6) in beagle puppies (age approximately 79 days). In the CPI2 and CAV2 groups, a lower respiratory illness developed by day 3 postinfection with clinical recovery by day 14. In the CAV2-CPI2 group, puppies were inoculated initially with CAV2 and 12 days later with CPI2. In this group, illness persisted until day 14 after infection with CPI2. Lung resistance (RL), dynamic (Cdyn) and static (Cst) lung compliance, functional residual capacity (FRC), and responsiveness to aerosolized histamine were measured before infection and at periodic intervals until 239 +/- 43 days of age. Lung function data were analyzed using a longitudinal random effects model. In all groups, FRC, Cst, and Cdyn increased with age. In all infected groups, the regression slopes for Cdyn were steeper than in controls. RL decreased linearly with age without group slope differences. Histamine reactivity increased with age, but there were no differences in slope among groups. Lung pathological studies showed areas of obliterative bronchiolitis and chronic small airways inflammation particularly in the CAV2 and CAV2-CPI2 groups. Thus, viral bronchiolitis produces chronic small airways inflammation in beagle puppies and alters the changes in lung function occurring with growth. Histamine reactivity increases with age and is not modified by viral infection.     

A note on the structure of system state spaces and its implications on the existence of non-repeatable experiments

The implication of state space structure on the existence of a repeatable experimentE designed to determine if a states∈L has propertyP or notP is investigated. It is shown that if a state spaceL is connected, then no experimentE is repeatable. This formalism is used to demonstrate that if a propertyP has an associated set of points inL which is dense with dense complement inL, then there exists no repeatable experimentE which can be used to test whethers has propertyP or notP. Other consequences of this formalization are discussed.     

Smoke aldehyde component influences pulmonary edema.

The pulmonary edema of smoke inhalation is caused by the toxins of smoke and not the heat. We investigated the potential of smoke consisting of carbon in combination with either acrolein or formaldehyde (both common components of smoke) to cause pulmonary edema in anesthetized sheep. Seven animals received acrolein smoke, seven animals received a low-dose formaldehyde smoke, and five animals received a high-dose formaldehyde smoke. Pulmonary arterial pressure, pulmonary capillary wedge pressure, and cardiac output were not affected by smoke in any group. Peak airway pressure increased after acrolein (14 +/- 1 to 21 +/- 2 mmHg; P less than 0.05) and after low- and high-dose formaldehyde (14 +/- 1 to 21 +/- 1 and 20 +/- 1 mmHg, respectively; both P less than 0.05). The partial pressure of O2 in arterial blood fell sharply after acrolein [219 +/- 29 to 86 +/- 9 (SE) Torr; P less than 0.05] but not after formaldehyde. Only acrolein resulted in a rise in lung lymph flow (6.5 +/- 2.2 to 17.9 +/- 2.6 ml/h; P less than 0.05). Lung lymph-to-plasma protein ratio was unchanged for all three groups, but clearance of lymph protein was increased after acrolein. After acrolein, the blood-free extravascular lung water-to-lung dry weight ratio was elevated (P less than 0.05) compared with both low- and high-dose formaldehyde groups (4.8 +/- 0.4 to 3.3 +/- 0.2 and 3.6 +/- 0.2, respectively). Lymph clearance (ng/h) of thromboxane B2, leukotriene B4, and the sulfidopeptide leukotrienes was elevated after acrolein but not formaldehyde.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasticity of the skeleton and skeletal deformities in zebrafish (Danio rerio) linked to rearing density

The teleost zebrafish (Danio rerio), an established model for human skeletal diseases, is reared under controlled conditions with defined parameters for temperature and photoperiod. Studies aimed at defining the proper rearing density have been performed with regard to behavioural and physiological stress response, sex ratio and reproduction. Studies concerning the effect of rearing density on the skeletal phenotype are lacking. This study analyses the response of the skeleton to different rearing densities and describes the skeletal deformities. Wild-type zebrafish were reared up to 30 dpf (days post-fertilization) in a common environment. From 30 to 90 dpf, animals were reared at three different densities: high density (HD), 32 fish l^–1; medium density (MD), 8 fish l^–1 and low density (LD), 2 fish l^–1. Animals at 30 and 90 dpf were collected and whole-mount stained with Alizarin red S to visualize mineralized tissues. The entire skeleton was analysed for meristic counts and 172 types of deformities. The results showed that the rearing density significantly influenced the specimens’ average standard length, which decreased with the increase in the rearing density. Differences in meristic counts among the three groups were not observed. Rearing density–independent malformations affected the ribs, neural arches and the spines of the abdominal region, as well as vertebrae of the caudal complex. The HD group showed the highest number of deformities per specimen, the highest number of observed types of deformities and, together with the MD group, the highest frequency of specimens affected by severe deformities. In particular, the HD group showed deformities affecting arches, spines and vertebral centra in the caudal region of the vertebral column. This study provides evidence of an effect of the rearing density on the development of different skeletal phenotypes.     

Regulation of growth by the trehalose pathway: Relationship to temperature and sucrose

Carbon signaling can override carbon supply in the regulation of growth. At least some of this regulation is imparted by the sugar signal trehalose 6-phosphate (T6P) through the protein kinase, SnRK1. This signaling pathway regulates biosynthetic processes involved in growth under optimal growing conditions. Recently, using a seedling system we showed that under sub-optimal conditions, such as cold, carbon signaling by T6P/ SnRK1 enables recovery of growth following relief of the stress. The T6P/ SnRK1 mechanism thus could be selected as a means of improving low temperature tolerance. High-throughput automated Fv/Fm measurements provide a potential means to screen for T6P/ SnRK1, and here we confirm through measurements of Fv/Fm in rosettes that T6P promotes low temperature tolerance and recovery during cold to warm transfer. Further, to better understand the coordination between sugars, trehalose pathway, and temperature-dependent growth, we examine the interrelationship between sugars, trehalose phosphate synthase (TPS), and trehalose phosphate phosphatase (TPP) gene expression and T6P content in seedlings. Sucrose, particularly when fed exogenously, correlated well with TPS1 and TPPB gene expression, suggesting that these enzymes are involved in maintaining carbon flux through the pathway in relation to sucrose supply. However, when sucrose accumulated to higher levels under low temperature and low N, TPS1 and TPPB expression were less directly related to sucrose; other factors may also contribute to regulation of TPS1 and TPPB expression under these conditions. TPPA expression was not related to sucrose content and all genes were not well correlated with endogenous glucose. Our work has implications for understanding acclimation to sink-limited growth conditions such as low temperature and for screening cold-tolerant genotypes with altered T6P/ SnRK1 signaling.     

CD40L-Tri, a novel formulation of recombinant human CD40L that effectively activates B cells

CD40L has a well-established role in enhancing the immunostimulatory capacity of normal and malignant B cells, but a formulation suitable for clinical use has not been widely available. Like other TNF family members, in vivo and in vitro activity of CD40L requires a homotrimeric configuration, and growing evidence suggests that bioactivity depends on higher-order clustering of CD40. We generated a novel formulation of human recombinant CD40L (CD40L-Tri) in which the CD40L extracellular domain and a trimerization motif are connected by a long flexible peptide linker. We demonstrate that CD40L-Tri significantly expands normal CD19+ B cells by over 20- to 30-fold over 14 days and induces B cells to become highly immunostimulatory antigen-presenting cells (APCs). Consistent with these results, CD40L-Tri-activated B cells could effectively stimulate antigen-specific T responses (against the influenza M1 peptide) from normal volunteers. In addition, CD40L-Tri could induce malignant B cells to become effective APCs, such that tumor-directed immune responses could be probed. Together, our studies demonstrate the potent immune-stimulatory effects of CD40L-Tri on B cells that enable their expansion of antigen-specific human T cells. The potent bioactivity of CD40L-Tri is related to its ability to self-multimerize, which may be facilitated by its long peptide linker.     

Draft genome sequence of Sinorhizobium meliloti RU11/001, a model organism for flagellum structure,motility and chemotaxis

Sinorhizobium meliloti of the order Rhizobiales is a symbiotic nitrogen-fixing bacterium nodulating plants of the genera Medicago, Trigonella and Melilotus and hence is of great agricultural importance. In its free-living state it is motile and capable of modulating its movement patterns in response to chemical attractants. Here, the draft genome consisting of a circular chromosome, the megaplasmids pSymA and pSymB and three accessory plasmids of Sinorhizobium meliloti RU11/001, a model organism for flagellum structure, motility and chemotaxis, is reported.     

Heat stress reveals a specialized variant of the pachytene checkpoint in meiosis of Arabidopsis thaliana

Plant growth and fertility strongly depend on environmental conditions such as temperature. Remarkably, temperature also influences meiotic recombination and thus, the current climate change will affect the genetic make-up of plants. To better understand the effects of temperature on meiosis, we followed male meiocytes in Arabidopsis thaliana by live cell imaging under three temperature regimes: at 21°C; at heat shock conditions of 30°C and 34°C; after an acclimatization phase of 1 week at 30°C. This work led to a cytological framework of meiotic progression at elevated temperature. We determined that an increase from 21°C to 30°C speeds up meiosis with specific phases being more amenable to heat than others. An acclimatization phase often moderated this effect. A sudden increase to 34°C promoted a faster progression of early prophase compared to 21°C. However, the phase in which cross-overs mature was prolonged at 34°C. Since mutants involved in the recombination pathway largely did not show the extension of this phase at 34°C, we conclude that the delay is recombination-dependent. Further analysis also revealed the involvement of the ATAXIA TELANGIECTASIA MUTATED kinase in this prolongation, indicating the existence of a pachytene checkpoint in plants, yet in a specialized form.

Live cell imaging of plants exposed to different heat stresses provides a temporal framework of meiosis at high temperatures in wild-type and mutants for several meiotic recombination factors.