Construction and characterization of avian Escherichia coli cya crp mutants

We constructed delta cya delta crp mutants of two avian septicemic Escherichia coli strains and evaluated their attenuation in virulence. The P1 phage was used to transfer cya::Tn10 from an E. coli K-12 strain into virulent avian O78 and O2 E. coli isolates. Tetracycline-resistant transductants were plated on Bochner-Maloy Medium, and tetracycline-sensitive colonies were selected, then tested by polymerase chain reaction to confirm that they had deletions of the cya gene. Deletions of crp were created by the same technique in isolates with deletions in cya. The delta cya and delta cya delta crp derivatives had slower growth rates, smaller colonies, and impaired fermentation of carbohydrates compared with their wild parents, and they did not revert. Attenuation of the mutant strains was evaluated by subcutaneous (s.c.) inoculation of day-old chicks and by intratracheal (i.t.) inoculation of 9-day-old chicks previously inoculated intranasally with infectious bronchitis virus. For the wild O78 strain and its delta cya and delta cya delta crp derivatives, the percentages of chicks that died within 6 days of s.c. injection of approximately 5 x 10(7) organisms were 100, 60, and 0, respectively. The corresponding percentages for wild-type O2 and its delta cya and delta cya delta crp mutants were 100, 70, and 20 at a dose of approximately 2 x 10(5) organisms. Following i.t. inoculation, group scores based on pathologic and bacteriologic findings were 51%, 15%, and 9% for wild, delta cya, and delta crp O78 strains (inoculum approximately 2 x 10(7) organisms) and 98%, 31%, and 11%, respectively, for the corresponding O2 strains (inoculum approximately 4 x 10(6) organisms). This study demonstrated reduced virulence and stability of the double mutant, which may useful as a live attenuated vaccine against poultry colibacillosis.     

Pharmacological characterization of human m1 muscarinic acetylcholine receptors with double mutations at the junction of TM VI and the third extracellular domain

A mutant human m5 receptor containing the mutations of Ser465 to Tyr and Thr466 to Pro showed constitutive activity. By replacing the equivalent Ser388 with Tyr and Thr389 with Pro, we created a mutant human m1 (Hm1) receptor with comparable double mutations. The mutant receptor, Hm1(Ser388Tyr, Thr389Pro), was stably expressed in A9 L cells and displayed enhanced responses to classical muscarinic agonists with significantly increased potencies. Choline, a normal component of growth media, showed an efficacy comparable to acetylcholine and carbachol at Hm1(Ser388Tyr, Thr389Pro) receptors. Methylcarbachol, a selective nicotinic agonist, exhibited partial agonist activity at human m1 wild-type receptors and full agonist activity at Hm1(Ser388Tyr, Thr389Pro) receptors. l-Hyoscyamine inhibited the activities of choline and methylcarbachol. Muscarinic antagonists displayed small reductions in binding affinities, although muscarinic agonists showed greatly increased binding affinities for Hm1(Ser388Tyr, Thr389Pro) receptors. All agonists, including choline and methylcarbachol, showed multiple affinity states at Hm1(Ser388Tyr, Thr389Pro) receptors in the absence of GppNHp. The high affinity binding sites for acetylcholine, arecoline and choline were shifted in the presence of GppNHp. These results suggest that Hm1(Ser388Tyr, Thr389Pro) is conformationally favorable for agonist binding and receptor activation.     

Inactivation of mycobacteriophage D29 using ferrous ammonium sulphate as a tool for the detection of viable Mycobacterium smegmatis and M. tuberculosis

There is still an urgent requirement for more sensitive, cost-effective methods for detection and susceptibility testing of mycobacteria in clinical samples. We have been investigating a simple bacteriophage-based system which could be used for both purposes. As this depends upon the detection of phages which have successfully infected cells, a key step is the efficient removal or inactivation of phages remaining free in the culture medium. We demonstrate here the use of ferrous ammonium sulphate as an effective agent for the inactivation of mycobacteriophage D29 without impairing phage replication in previously infected host bacteria. Using this property, we report the detection of viable Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis using simple low-cost technology. The method is highly sensitive, since it is able to detect 10 colony-forming units of M. smegmatis. It is also rapid, with the detection of M. tuberculosis in sputum specimens within 48 h.     

Pulpal response to a resin-modified glass-ionomer material on nonexposed and exposed monkey pulps

OBJECTIVE: This study evaluated the biocompatibility of a resin-modified glass-ionomer material on monkey pulps. METHOD AND MATERIALS: Standardized Class V cavities were prepared in 112 teeth distributed in six healthy adult monkeys. The resin-modified glass-ionomer cement was placed in 24 nonexposed and 36 exposed pulps according to manufacturer's instructions. ZOE was used as a control in nonexposed pulps, while calcium hydroxide was used as a control for exposed pulps. Tissues were collected at 6 to 7, 21 to 27, and 90 to 97 days. After demineralization, the teeth were serially sectioned, stained, and observed by light microscopy. RESULTS: Except for one resin-modified glass-ionomer pulp at 6 days, there were no differences between the responses of nonexposed pulps to resin-modified glass-ionomer specimens and ZOE controls. In exposed pulps, eight of 36 resin-modified glass-ionomer pulps showed various grades of inflammatory response, all associated with stained bacteria. Pulpal healing was similar in both resin-modified glass-ionomer and calcium hydroxide direct-capped exposures. Twenty-two of 26 exposed pulps restored with the resin-modified glass-ionomer cement showed dentin bridge formation at 21 and 97 days. CONCLUSION: The resin-modified glass-ionomer material exhibited acceptable biologic compatibility in exposed and nonexposed cavities.     

The anatomy of a hypoxic operator in Saccharomyces cerevisiae

Aerobic repression of the hypoxic genes of Saccharomyces cerevisiae is mediated by the DNA-binding protein Rox1 and the Tup1/Ssn6 general repression complex. To determine the DNA sequence requirements for repression, we carried out a mutational analysis of the consensus Rox1-binding site and an analysis of the arrangement of the Rox1 sites into operators in the hypoxic ANB1 gene. We found that single base pair substitutions in the consensus sequence resulted in lower affinities for Rox1, and the decreased affinity of Rox1 for mutant sites correlated with the ability of these sites to repress expression of the hypoxic ANB1 gene. In addition, there was a general but not complete correlation between the strength of repression of a given hypoxic gene and the compliance of the Rox1 sites in that gene to the consensus sequence. An analysis of the ANB1 operators revealed that the two Rox1 sites within an operator acted synergistically in vivo, but that Rox1 did not bind cooperatively in vitro, suggesting the presence of a higher order repression complex in the cell. In addition, the spacing or helical phasing of the Rox1 sites was not important in repression. The differential repression by the two operators of the ANB1 gene was found to be due partly to the location of the operators and partly to the sequences between the two Rox1-binding sites in each. Finally, while Rox1 repression requires the Tup1/Ssn6 general repression complex and this complex has been proposed to require the aminoterminal regions of histones H3 and H4 for full repression of a number of genes, we found that these regions were dispensable for ANB1 repression and the repression of two other hypoxic genes.     

Formaldehyde in cryoprotectant propanediol and effect on mouse zygotes

Cryopreservation of human zygotes and embryos has been routinely performed by in-vitro fertilization clinics for many years. Karran and Legge (1996) first reported that formaldehyde (FA) present in the cryoprotective solutions can have a deleterious effect on mouse oocytes. FA is a cytotoxic, carcinogenic and mutagenic chemical. The effect of FA on mouse zygotes was investigated. In addition, the concentrations of FA in propanediol (PROH) obtained from various sources were determined. Pooled 1-cell embryos were dispensed into droplets of modified Ham's F10 or human tubal fluid containing various concentrations of FA. Since bovine serum albumin (BSA) may minimize toxicity additional trials were done as above in the absence of BSA. FA concentration in the standard 1.5 M PROH, from different sources in water, was measured in the same assay using a standard curve of 0-100 microM FA. FA in a complex medium had a significant deleterious effect on embryo development and hatching but only at 1 mM concentration (P < 0.000001; see Tables I-III). There was no significant effect of FA at 100 microM. However, in a simple medium even 50 microM FA decreased embryo hatching. FA was present in 1.5 M PROH from different sources (range 1.0-35.3 microM concentration). It appears that FA concentrations do not increase with storage because FA concentrations were low even after opening and storage for 3 years on the shelf. This suggests that FA is a contaminant during the manufacturing process and may vary from manufacturer to manufacturer and batch to batch. Until further studies are done to confirm the lack of toxicity to embryos during cryopreservation (with or without FA scavengers) it may be prudent to screen all batches of cryoprotectants for FA as part of quality control.     

Therapists' self-reported training and success rates in treating clients with childhood sexual abuse

In this two-part study, three groups of therapists (16 psychologists, 11 counselors, and 14 social workers) were compared with regard to their success rates with clients presenting with childhood sexual abuse, and 43 graduate programs were asked whether they offered courses covering counseling techniques with such clients. Therapists' self-reported success rates were not significantly related to therapists' training, but type of therapy was. Given a response rate of 18.6%, self-reports should be treated cautiously. Over two-thirds of the training programs (45% response rate) offered courses covering issues in childhood sexual abuse and 67% offered courses covering counseling techniques with such clients. However, in 85% of these courses, treatment of childhood sexual abuse was a topic covered under more general course headings.     

The risk of acquiring Q fever on farms: a seroepidemiological study

OBJECTIVES: To determine the occupational risk of Q fever. DESIGN: Cohort study. SETTING: Community: five English local authority districts. SUBJECTS AND METHODS: Prevalence and incidence of immunoglobulin G (IgG) specific antibody to Coxiella burnetii phase II antigen was measured in a representative (study) cohort of farm workers in the United Kingdom, and detailed exposure data were collected. Also seroprevalence of Q fever in a (control) cohort of police and emergency service personnel was measured. RESULTS: Prevalence was significantly (P < 0.01) higher in the study cohort (105/385 v 43/395). During the first 12 month period after enrollment no seroconversions were found (upper 95% confidence limit: 1318/100,000/year). During the second 12 month period after enrollment two seroconversions were found, equalling an incidence of 813/100,000/year (95% confidence interval (95% CI) 98-2937/100,000/year). No association was found between seroprevalence and age. In the study cohort, extent of total contact with farm animals seemed more important than exposure to any specific animal: full time employees were more than four times more likely to be antibody positive than part time employees (P < 0.05). Exposure to cattle, but not sheep, goats, cats, raw milk, and hay (all reported sources of Q fever) was associated with being positive to Coxiella burnetii IgG by univariate analysis but this association was not independent of total farm animal contact. CONCLUSIONS: The risk of Q fever on livestock farms is related to contact with the farm environment rather than any specific animal exposure. The absence of an increasing prevalence with age suggests that exposure may occur as clusters in space and time (outbreaks).     

Inhibition of platelet function by A02131-1, a novel inhibitor of cGMP-specific phosphodiesterase, in vitro and in vivo

The effect of A02131-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzyl thieno (3,2-c)pyrazole], a cGMP-specific phosphodiesterase (PDE) inhibitor, on platelet function was investigated. The compound was found to inhibit the aggregation of and adenosine triphosphate (ATP) release from human platelet-rich plasma and washed platelets that were induced by aggregation inducing drugs such as arachidonic acid (AA), collagen, U46619, platelet-activating factor (PAF), adenosine diphosphate (ADP) and A23187, and the inhibitory effect was concentration-dependent. A02131-1 also disaggregated the performed platelet aggregates induced by these inducers. Thromboxane B2 (TXB2) formations caused by collagen, PAF, ADP, and A23187 were inhibited by A02131-1 at concentrations that did not affect the AA-induced formation of TXB2 and prostaglandin D2 (PGD2). A02131-1 suppressed both the generation of inositol 1,4,5-triphosphate (IP3) and the increase of intracellular Ca2+ concentration stimulated by these aggregation inducers. A02131-1 was shown to increase the cAMP and cGMP levels in platelets and the extent was found to be dependent on concentration as well as time. A02131-1 increased the cAMP level much more slowly than the cGMP level. Activities of adenylate cyclase, guanylate cyclase, and PDEs (type I and III) were not altered by A02131-1. However, the activity of cGMP-specific PDE (type V) was inhibited by A02131-1. The antiplatelet aggregation activity and the effect on raising cAMP level of A02131-1 were both potentiated by prostaglandin E1 (PGE1). In the mouse tail bleeding test, A02131-1 was clearly shown to be more effective than dipyridamole in prolonging the tail bleeding time of conscious mice. These data indicate that A02131-1 is a cGMP-specific PDE (type V) inhibitor in human platelets.