OmpF enhances the ability of BtuB to protect susceptible Escherichia coli cells from colicin E9 cytotoxicity

The outer membrane (OM) vitamin B(12) receptor, BtuB, is the primary receptor for E group colicin adsorption to Escherichia coli. Cell death by this family of toxins requires the OM porin OmpF but its role remains elusive. We show that OmpF enhances the ability of purified BtuB to protect bacteria against the endonuclease colicin E9, demonstrating either that the two OM proteins form the functional receptor or that OmpF is recruited for subsequent translocation of the bacteriocin. While stable binary colicin E9-BtuB complexes could be readily shown in vitro, OmpF-containing complexes could not be detected, implying that OmpF association with the BtuB-colicin complex, while necessary, must be weak and/or transient in nature.     

Mechanistic considerations in small fish carcinogenicity testing

Historically, small fish species have proven useful both as environmental sentinels and as versatile test animals in toxicity and carcinogenicity bioassays. They can be bred in large numbers, have low maintenance and bioassay costs, and have a low background incidence of tumors. However, more mechanistic information is needed to help validate the information garnered from these models and to keep pace with other more fully developed animal models. This paper focuses on mechanistic considerations when using small fish models for carcinogenicity testing. Several small aquarium fish species have proven useful. The Japanese medaka is perhaps the best characterized small fish model for carcinogenicity testing; however, the zebrafish is emerging as an important model because it is well characterized genetically. Both route and methodology of exposure may affect the outcome of the study. Most studies have been conducted by introducing the test compound into the ambient water, but dietary exposures and embryo microinjection have also been used. Other considerations in study design include use of an initiating carcinogen, such as diethlynitrosamine, and differences in xenobiotic metabolism, such as the fact that fish CYP2B is refractory to phenobarbital induction. The small size of these models has perhaps limited some types of mechanistic studies, such as formation and repair of DNA adducts in response to carcinogen exposure. However, improved analytical methods are allowing greater resolution and should be applied to small fish species. Slide-based methods such as immunohistochemistry are an important adjunct to routine histopathology and should be included in study design. However, there is a need for development of more species-specific antibodies for fish research. There is also a need for more fish-specific data on cytokines, serum biochemistry, and oncogenes to strengthen the use of these important test models.     

Inhibiting adenosine deaminase modulates the systemic inflammatory response syndrome in endotoxemia and sepsis

By pharmacological manipulation of endogenous adenosine, using chemically distinct methods, we tested the hypothesis that endogenous adenosine tempers proinflammatory cytokine responses and oxyradical-mediated tissue damage during endotoxemia and sepsis. Rats were pretreated with varying doses of pentostatin (PNT; adenosine deaminase inhibitor) or 8-sulfophenyltheophylline (8-SPT; adenosine receptor antagonist) and then received either E. coli endotoxin (lipopolysaccharide; 0.01 or 2.0 mg/kg) or a slurry of cecal matter in 5% dextrose in water (200 mg/kg). Resultant levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-10 were measured in serum and in liver and spleen. Untreated, 2 mg/kg lipopolysaccharide elevated serum TNF-alpha, IL-1beta, and IL-10. PNT dose dependently attenuated, without ablating, the elevation in serum TNF-alpha and IL-1beta and raised liver and spleen IL-10. PNT also attenuated elevation of TNF-alpha in serum, liver, and spleen at 4 and 24 h after sepsis induction, and 8-SPT resulted in higher proinflammatory cytokines. Modulating endogenous adenosine was also effective in exacerbated (8-SPT) or diminished (PNT) tissue peroxidation. Survival from sepsis was also improved when PNT was used as a posttreatment. These data indicate that endogenous adenosine is an important modulatory component of systemic inflammatory response syndromes. These data also indicate that inhibition of adenosine deaminase may be a novel and viable therapeutic approach to managing the systemic inflammatory response syndrome without ablating important physiological functions.     

Knockdown of connexin43-mediated regulation of the zone of polarizing activity in the developing chick limb leads to digit truncation

In the developing chick wing, the use of antisense oligodeoxynucleotides to transiently knock down the expression of the gap junction protein, connexin43 (Cx43), results in limb patterning defects, including deletion of the anterior digits. To understand more about how such defects arise, the effects of transient Cx43 knockdown on the expression patterns of several genes known to play pivotal roles in limb formation were examined. Sonic hedgehog (Shh), which is normally expressed in the zone of polarizing activity (ZPA) and is required to maintain both the ZPA and the apical ectodermal ridge (AER), was found to be downregulated in treated limbs within 30 h. Bone morphogenetic protein-2 (Bmp-2), a gene downstream of Shh, was similarly downregulated. Fibroblast growth factor-8 expression, however, was unaltered 30 h after treatment but was greatly reduced at 48 h post-treatment, when the AER begins to regress. Expressions of Bmp-4 and Muscle segment homeobox-like gene (Msx-1) were not affected at any of the time points examined. Cx43 expression is therefore involved in some, but not all patterning cascades, and appears to play a role in the regulation of ZPA activity.     

Dynamics of T cells and TCR excision circles differ after treatment of acute and chronic HIV infection

We quantified T cell proliferation and thymic function in primary HIV infection (PHI; n = 19) and chronic HIV infection (CHI; n = 14) by measuring Ki67 staining and TCR excision circle (TREC) number. After antiretroviral therapy of PHI there is a profound decrease in the number and percentage of Ki67(+) T cells (<6% Ki67(+)) with no significant increase in TREC per million cells and a transient increase in TREC per milliliter. In contrast, after antiretroviral therapy of CHI there is a reduction in the percentage but little change in the total number of Ki67(+)CD4(+) T cells associated with increases in both TREC per million cells and TREC per milliliter. Using a mathematical model that accounts for proliferation, death, and redistribution of T cells, we find that redistribution is consistent with the TREC changes observed during treatment of PHI and that an increase in thymic output is needed to explain the increase in TREC during treatment of CHI. Consideration of TREC per milliliter shows that changes in proliferation alone cannot explain the changes in TREC. In addition, although increased proliferation of memory cells in HIV infection has been established, we find no difference in TREC per million CD45RA(-) "memory" T cells between healthy and infected individuals (p = 0.154 for CD4(+); p = 0.383 for CD8(+)). Finally, although the number of TREC per million cells is always much lower in memory T cells than in naive T cells, in the setting of HIV infection, given that memory cells make up a larger proportion of total T cells, we find that 50% of TREC per milliliter in CD4(+) T cells is harbored in the CD45RA(-) "memory" subset of our infected subjects.     

Silver enhances stamen development in female white campion (Silene latifolia [Caryophyllaceae])

Sex expression in the dioecious plant white campion (Silene latifolia Poiret subsp. alba) appears to be insensitive to exogenous applications of auxins, cytokinins, gibberellic acid, and ethylene; however, silver thiosulfate (Ag(2)S(2)O(3)), an ethylene inhibitor, enhanced stamen development in female white campion. In wild-type females, stamen development is arrested before the microspore mother cells are formed. In contrast, stamens of Ag(2)S(2)O(3)-treated females completed meiosis and produced microspores. Stamen development for these females was incomplete, however, and pollen did not mature. Ag(2)S(2)O(3) stimulated stamen development to the same extent in asexual white campion mutants that retained a Y chromosome but had lost Y-linked genes needed for early stages of stamen development. Although Ag(2)S(2)O(3) can inhibit ethylene signaling, the enhancement of stamen development in female white campion cannot be explained as a loss of ethylene response because no other ethylene inhibitor tested (1-methylcyclopropene, trans-cyclooctene, aminoethoxyvinylglycine, and cobalt chloride) caused stamens to develop in female plants. In addition, application of other metal ions could not enhance stamen development. Therefore, the effect we observed on female white campion was specifically caused by silver ions but not by their action on ethylene signaling.     

Water-soluble fluorescent diblock nanospheres

The hydroxyl groups of poly(tert-butyl acrylate)-block-poly(2-hydroxyethyl methacrylate) or PtBA-b-PHEMA were reacted with succinic anhydride to introduce some carboxyl groups into the PHEMA block. Such carboxyl groups were then reacted with Texas-red cadverine (TX-NH(2)) to incorporate dye molecules. The TX-bearing diblocks formed probably spherical micelles in block-selective solvent DMF/toluene containing 2% DMF. "Permanent" micelles or nanospheres were prepared after cross-linking the TX-bearing PHEMA core block. Such nanospheres were made water soluble by cleaving the tert-butyl groups from the PtBA coronas. Water-soluble nanospheres with high TX numbers and fluorescence quantum yields may find applications in fluorescence in situ hybridization assays.     

A high-throughput Arabidopsis reverse genetics system

A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymmetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from approximately 100000 transformed lines. A total of 85108 TAIL-PCR products from 52964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org.     

Rectal and colonic distension elicit viscerovisceral reflexes in humans

Colonic transit is slowed in patients with disordered rectal evacuation, but the mechanism of this phenomenon is unclear. Our objective was to investigate rectocolonic inhibitory reflexes in humans to provide potential insight into patients with obstructed defecation. In 30 healthy subjects, a barostat-manometric assembly recorded colonic tone and phasic activity in the descending colon during rectal distension and recorded rectal tone during colonic distension. Phasic distensions were 8, 16, and 32 mmHg above balloon operating pressure, and staircase inflations were comprised of balloon inflation then deflation in 2-mmHg increments at 30-s intervals from 0 to 36 mmHg. Colonic balloon volumes increased to a similar extent during phasic rectal distensions 8, 16, and 32 mmHg above operating pressure, reflecting reduced colonic tone; balloon volumes also increased and phasic pressure activity decreased during staircase rectal distensions. In contrast, rectal balloon volume declined, reflecting increased tone during phasic and staircase colonic distensions. Thus rectal distension inhibited colonic motor activity, indicative of a viscerovisceral inhibitory reflex.     

Stimulation of chloride secretion by baicalein in isolated rat distal colon

The effect of baicalein on mucosal ion transport in the rat distal colon was investigated in Ussing chambers. Mucosal addition of baicalein (1-100 microM) elicited a concentration-dependent short-circuit current (I(sc)) response. The increase in I(sc) was mainly due to Cl(-) secretion. The presence of mucosal indomethacin (10 microM) significantly reduced both the basal and subsequent baicalein-evoked I(sc) responses. The baicalein-induced I(sc) were inhibited by mucosal application of diphenylamine-2-carboxylic acid (100 microM) and glibenclamide (500 microM) and basolateral application of chromanol 293B (30 microM), a blocker of K(v)LQT1 channels and Ba(2+) ions (5 mM). Treatment of the colonic mucosa with baicalein elicited a threefold increase in cAMP production. Pretreating the colonic mucosa with carbachol (100 microM, serosal) but not thapsigargin (1 microM, both sides) abolished the baicalein-induced I(sc). Addition of baicalein subsequent to forskolin induced a further increase in I(sc). These results indicate that the baicalein evoked Cl(-) secretion across rat colonic mucosa, possibly via a cAMP-dependent pathway. However, the action of baicalein cannot be solely explained by its cAMP-elevating effect. Baicalein may stimulate Cl(-) secretion via a cAMP-independent pathway or have a direct effect on cystic fibrosis transmembrane conductance regulator.