Alternative mechanism for bacteriophage adsorption to the motile bacterium Caulobacter crescentus

2D and 3D cryo-electron microscopy, together with adsorption kinetics assays of ϕCb13 and ϕCbK phage-infected Caulobacter crescentus, provides insight into the mechanisms of infection. ϕCb13 and ϕCbK actively interact with the flagellum and subsequently attach to receptors on the cell pole. We present evidence that the first interaction of the phage with the bacterial flagellum takes place through a filament on the phage head. This contact with the flagellum facilitates concentration of phage particles around the receptor (i.e., the pilus portals) on the bacterial cell surface, thereby increasing the likelihood of infection. Phage head filaments have not been well characterized and their function is described here. Phage head filaments may systematically underlie the initial interactions of phages with their hosts in other systems and possibly represent a widespread mechanism of efficient phage propagation

Neurotransmitter organization of the nucleus of Edinger-Westphal and its projection to the avian ciliary ganglion

Two morphologically distinct types of preganglionic endings are observed in the avian ciliary ganglion: boutonal and cap-like. Boutonal endings synapse on ciliary ganglion neurons (called choroidal neurons) innervating choroidal blood vessels, while cap-like endings synapse on ciliary ganglion neurons (called ciliary neurons) controlling the lens and pupil. Some of both types of preganglionic endings contain the neuropeptides substance P (SP) and/or leucine-enkephalin (LENK). Although both types of preganglionic terminals are also known to be cholinergic, there has been no direct evidence that SP and LENK are found in cholinergic endings in the ciliary ganglion. The present studies in pigeons, which involved the use of single- and double-label immunohistochemical techniques, were undertaken to examine this issue, as well as to (1) determine the relative percentages of the boutonal and cap-like endings that contain SP, LENK, or both SP and LENK; and (2) determine if the two different types of terminals in the ciliary ganglion arise from different subdivisions of the nucleus of Edinger-Westphal (EW). Single- and double-label immunohistochemical studies revealed that all neurons of EW, regardless of whether they contained immunohistochemically detectible amounts of SP or LENK, are cholinergic. In the medial subdivision of EW (EWM), which was found to contain approximately 700 neurons, 20.2% of these neurons were observed to contain both SP and LENK, while 11.6% were observed to contain SP only and 10.7% were observed to contain LENK only. In contrast, in lateral EW (EWL), which was found to contain approximately 500 neurons, 16.2% of the neurons were observed to contain both SP and LENK, while 19.2% of the neurons were observed to contain SP only and 12.6% were observed to contain LENK only. Retrograde-labeling studies involving horseradish peroxidase injections into the ciliary ganglion revealed that EW was the sole source of input to the ciliary ganglion and all, or nearly all, neurons in EW innervate the ciliary ganglion. Immunohistochemical labeling of the ciliary ganglion neurons with an antiserum against choline acetyltransferase revealed that approximately 900 choroidal neurons and approximately 600 ciliary neurons are present in the ganglion, all of which receive cholinergic preganglionic endings. Of the choroidal neurons, 94% receive butonal terminals containing both SP and LENK, while only 2% receive SP+ only boutonal endings and 2% receive LENK+ only butonal endings. Of the ciliary neurons, 25% receive cap-like endings containing both SP and LENK, 30% receive cap-like endings containing only SP and 3% receive cap-like endings containing only LENK. Total unilateral lesions of EW resulted in the loss of all SP+ or LENK+ terminals in the ipsilateral ganglion. Subtotal EW lesions that spared either part of EWM or part of EWL revealed that boutonal endings arise from EWM neurons and cap-like endings from EWL neurons. The present results suggest that the choroidal neurons, which regulate choroidal blood flow, may be relatively uniform in their functional properties since they nearly all receive boutonal endings from EWM that co-contain SP, LENK, and acetylcholine. In contrast, the ciliary neurons, which receive their preganglionic input from EWL, may consist of at least three major functionally distinct subgroups: (1) those receiving SP/LENK/acetylcholine-containing cap-like endings; (2) those receiving SP/acetylcholine-containing cap-like endings; and (3) those receiving acetylcholine-containing cap-like endings. The functional diversity of ciliary neurons may in part be related to the fact that some ciliary neurons innervate the iris and others the ciliary body

Investigate a Gas Well Performance Using Nodal Analysis

Gas condensate well has unique reservoir characteristics and ups and downs in well behaviour affect the production rate significantly. A proper optimization can reduce the operating cost, maximize the hydrocarbon recovery and increase the net present value. Well level optimization can be achieved through optimizing well parameters, such as wellhead, tubing size, and skin factor. All of these factors have been investigated using a real field of Thrace Basin and PROSPER simulation program. The history matching data are validated to identify the future performance prediction for the same reservoir deliverability following the period changes. Therefore, predicted results are compared and validated with measured field data to provide the best production practices. Moreover, the results show that the skin factor has a large influence on the production rate by 45% reduction. The reduction in the reservoir pressure declines the production rate dramatically resulted in 70% decline. While manipulating the wellhead pressure shows minor decline compare to tubing size that does not show any significant change to production rate

Nongenomic oestrogen signalling in oestrogen receptor negative breast cancer cells: a role for the angiotensin II receptor AT1

INTRODUCTION: Oestrogens can mediate some of their cell survival properties through a nongenomic mechanism that involves the mitogen-activated protein kinase (MAPK) pathway. The mechanism of this rapid signalling and its dependence on a membrane bound oestrogen receptor (ER), however, remains controversial. The role of G-protein-coupled receptor and epidermal growth factor (EGF) receptor in an ER-independent signalling pathway modulated by oestrogen was investigated. METHODS: ER-positive and ER-negative breast cancer cell lines (MCF-7 and SKBR3) and primary breast cancer cell cultures were used in this study. Cell proliferation was assessed using standard MTT assays. Protein and cAMP levels were detected by Western blotting and ELISA, respectively. Antigen localization was performed by immunocytochemistry, immunohistochemistry and immunofluorescence. Protein knockdown was achieved using small interfering RNA technologies. RESULTS: EGF and oestrogen, alone and in combination, induced cell proliferation and phosphorylation of MAPK proteins Raf and ERK (extracellular signal regulated kinase)1/2 in both ER-negative SKBR3 and ER-positive MCF-7 human breast cancer cell lines. Increased Raf phosphorylation was also observed in primary human breast cultures derived from ER-positive and ER-negative breast tumours. Oestrogen induced an increase in intracellular cAMP in ER-negative SKBR3 human breast cancer cells. Oestrogen-mediated cell growth and phosphorylation of MAPK was modified by the EGF receptor antagonist AG1478, the G-protein antagonist pertussis toxin, and the angiotensin II receptor antagonist saralasin. Knockdown of angiotensin II type 1 receptor (AT1) protein expression with small interfering RNA attenuated oestrogen-induced Raf phosphorylation in ER-negative cells. AT1 receptor was found to be expressed in the cell membrane of breast tumour epithelial cells. CONCLUSION: These findings provide evidence that, in breast cancer cells, oestrogen can signal through AT1 to activate early cell survival mechanisms in an ER-independent manner

Screen for Localized Proteins in Caulobacter crescentus

Precise localization of individual proteins is required for processes such as motility, chemotaxis, cell-cycle progression, and cell division in bacteria, but the number of proteins that are localized in bacterial species is not known. A screen based on transposon mutagenesis and fluorescence activated cell sorting was devised to identify large numbers of localized proteins, and employed in Caulobacter crescentus. From a sample of the clones isolated in the screen, eleven proteins with no previously characterized localization in C. crescentus were identified, including six hypothetical proteins. The localized hypothetical proteins included one protein that was localized in a helix-like structure, and two proteins for which the localization changed as a function of the cell cycle, suggesting that complex three-dimensional patterns and cell cycle-dependent localization are likely to be common in bacteria. Other mutants produced localized fusion proteins even though the transposon has inserted near the 5′ end of a gene, demonstrating that short peptides can contain sufficient information to localize bacterial proteins. The screen described here could be used in most bacterial species

Genetic and Computational Identification of a Conserved Bacterial Metabolic Module

We have experimentally and computationally defined a set of genes that form a conserved metabolic module in the α-proteobacterium Caulobacter crescentus and used this module to illustrate a schema for the propagation of pathway-level annotation across bacterial genera. Applying comprehensive forward and reverse genetic methods and genome-wide transcriptional analysis, we (1) confirmed the presence of genes involved in catabolism of the abundant environmental sugar myo-inositol, (2) defined an operon encoding an ABC-family myo-inositol transmembrane transporter, and (3) identified a novel myo-inositol regulator protein and cis-acting regulatory motif that control expression of genes in this metabolic module. Despite being encoded from non-contiguous loci on the C. crescentus chromosome, these myo-inositol catabolic enzymes and transporter proteins form a tightly linked functional group in a computationally inferred network of protein associations. Primary sequence comparison was not sufficient to confidently extend annotation of all components of this novel metabolic module to related bacterial genera. Consequently, we implemented the Graemlin multiple-network alignment algorithm to generate cross-species predictions of genes involved in myo-inositol transport and catabolism in other α-proteobacteria. Although the chromosomal organization of genes in this functional module varied between species, the upstream regions of genes in this aligned network were enriched for the same palindromic cis-regulatory motif identified experimentally in C. crescentus. Transposon disruption of the operon encoding the computationally predicted ABC myo-inositol transporter of Sinorhizobium meliloti abolished growth on myo-inositol as the sole carbon source, confirming our cross-genera functional prediction. Thus, we have defined regulatory, transport, and catabolic genes and a cis-acting regulatory sequence that form a conserved module required for myo-inositol metabolism in select α-proteobacteria. Moreover, this study describes a forward validation of gene-network alignment, and illustrates a strategy for reliably transferring pathway-level annotation across bacterial species

Phosphate Starvation Triggers Production and Secretion of an Extracellular Lipoprotein in Caulobacter crescentus

Life in oligotrophic environments necessitates quick adaptive responses to a sudden lack of nutrients. Secretion of specific degradative enzymes into the extracellular medium is a means to mobilize the required nutrient from nearby sources. The aquatic bacterium Caulobacter crescentus must often face changes in its environment such as phosphate limitation. Evidence reported in this paper indicates that under phosphate starvation, C. crescentus produces a membrane surface-anchored lipoprotein named ElpS subsequently released into the extracellular medium. A complete set of 12 genes encoding a type II secretion system (T2SS) is located adjacent to the elpS locus in the C. crescentus genome. Deletion of this T2SS impairs release of ElpS in the environment, which surprisingly remains present at the cell surface, indicating that the T2SS is not involved in the translocation of ElpS to the outer membrane but rather in its release. Accordingly, treatment with protease inhibitors prevents release of ElpS in the extracellular medium suggesting that ElpS secretion relies on a T2SS-secreted protease. Finally, secretion of ElpS is associated with an increase in alkaline phosphatase activity in culture supernatants, suggesting a role of the secreted protein in inorganic phosphate mobilization. In conlusion, we have shown that upon phosphate starvation, C. crescentus produces an outer membrane bound lipoprotein, ElpS, which is further cleaved and released in the extracellular medium in a T2SS-dependent manner. Our data suggest that ElpS is associated with an alkaline phosphatase activity, thereby allowing the bacterium to gather inorganic phosphates from a poor environment

Asthma Pregnancy Alters Postnatal Development of Chromaffin Cells in the Rat Adrenal Medulla

Background: Adrenal neuroendocrine plays an important role in asthma. The activity of the sympathoadrenal system could be altered by early life events. The effects of maternal asthma during pregnancy on the adrenal medulla of offspring remain unknown. Methodology/Principal Findings: This study aims to explore the influence of maternal asthma during pregnancy on the development and function of adrenal medulla in offspring from postnatal day 3 (P3) to postnatal day 60 (P60). Asthmatic pregnant rats (AP), nerve growth factor (NGF)-treated pregnant rats (NP) and NGF antibody-treated pregnant rats (ANP) were sensitized and challenged with ovalbumin (OVA); NP and ANP were treated with NGF and NGF antibody respectively. Offspring rats from the maternal group were divided into four groups: offspring from control pregnant rats (OCP), offspring from AP (OAP), offspring from NP (ONP), and offspring from ANP (OANP). The expressions of phenylethanolamine N-methyltransferase (PNMT) protein in adrenal medulla were analyzed. The concentrations of epinephrine (EPI), corticosterone and NGF in serum were measured. Adrenal medulla chromaffin cells (AMCC) were prone to differentiate into sympathetic nerve cells in OAP and ONP. Both EPI and PNMT were decreased in OAP from P3 to P14, and then reached normal level gradually from P30 to P60, which were lower from birth to adulthood in ONP. Corticosterone concentration increased significantly in OAP and ONP. Conclusion/Significance: Asthma pregnancy may promote AMCC to differentiate into sympathetic neurons in offspring rats and inhibit the synthesis of EPI, resulting in dysfunction of bronchial relaxation