Effect of protein kinase C inhibitors and activators on corneal re-epithelialization in the rat

PURPOSE: To examine the ability of protein kinase C (PKC) inhibitors and activators to influence the rate of corneal re-epithelialization in the rat. METHOD: Rat corneas with 3 mm diameter central epithelial abrasions were organ-cultured in control medium or in medium with inhibitors or activators of PKC. RESULTS: In control corneas, the defect was completely re-epithelialized by 25 hr. In the presence of the PKC inhibitors staurosporine (100 nM), sphinganine (50 mumol/l), or H-7 (100 mumol/l) there were significantly larger epithelial defects than in controls after 5-25 hr of incubation. Re-epithelialization rates were similar to control corneas when the incubation medium contained HA1004 (100 mumol/l), an analogue of H-7 that is a potent inhibitor of cyclic adenosine monophosphate- and cyclic guanosine monophosphate-dependent protein kinases and a weak inhibitor of PKC. Two PKC activators, 1-oleoyl-2-acetyl-sn-glycerol (OAG) and phorbol 12-myristate 13-acetate (PMA), were unable to enhance the rate of epithelial wound healing. CONCLUSIONS: Our results suggest that PKC activity is an important factor in regulating corneal epithelial wound healing, presumably by influencing cell migration. Moreover, the results with OAG and PMA suggest that PKC is maximally activated during re-epithelialization in this organ-culture assay.     

Fentanyl and its analogs desensitize the cloned mu opioid receptor

Fentanyl, and its structural analogs lofentanil and sufentanil, are potent analgesics used clinically in the management of pain. However, the high analgesic potency of these compounds is limited by the development of tolerance after chronic use. To investigate whether their tolerance development may be related to mu receptor desensitization, the cloned mouse mu receptor as well as mutant forms of the receptor were stably expressed in HEK 293 cells and tested for their response to continuous opioid treatment. Fentanyl and its analogs potently bound to the mu receptor and effectively inhibited cAMP accumulation. Three-hour pretreatment of mu receptors with fentanyl and its analogs desensitized the mu receptor by uncoupling it from adenylyl cyclase. The fentanyl analogs caused a slight internalization of the mu receptor as accessed by antibody binding to the epitope-tagged mu receptor. Truncation of the mu receptor by removal of its carboxyl terminus at Glu341 did not affect the ability of the fentanyl analogs to bind to and activate the mu receptor nor did it prevent the fentanyl analogs from desensitizing the receptor. In a previous study we showed that morphine did not desensitize the cloned mu receptor even though it is a potent and effective agonist at the mu receptor. Mutagenesis studies revealed that morphine interacts differently with the mu receptor to activate it than do the fentanyl analogs which may explain its lack of desensitization of the mu receptor. These results indicate that desensitization of the mu receptor may be a molecular basis for the development of tolerance to fentanyl and its analogs.     

Escherichia coli O157:H7 requires intimin for enteropathogenicity in calves

Enterohemorrhagic Escherichia coli (EHEC) strains require intimin to induce attaching and effacing (A/E) lesions in newborn piglets. Infection of newborn calves with intimin-positive or intimin-negative EHEC O157:H7 demonstrated that intimin is needed for colonization, A/E lesions, and disease in cattle. These results suggest that experiments to determine if intimin-based vaccines reduce O157:H7 levels in cattle are warranted.     

Chromosomal aberrations, sister-chromatid exchanges, cells with high frequency of SCE, micronuclei and comet assay parameters in 1, 3-butadiene-exposed workers

The association of occupational exposure to 1,3-butadiene (BD) and induction of cytogenetic damage in peripheral lymphocytes was studied in 19 male workers from a monomer production unit and 19 control subjects from a heat production unit. The exposure to BD was measured by passive personal monitors. The following biomarkers were used: chromosomal aberrations (CA), sister chromatid exchanges (SCE), cells with a high frequency of SCE (HFC), micronuclei, comet assay parameters like tail length (TL) and percentage of DNA in tail [T (%)] and polymorphisms of GSTM1 and GSTT1 genotypes. BD exposure with a median value of 0.53 mg/m3 (range: 0.024-23.0) significantly increased (a) the percentage of cells with chromosomal aberrations in exposed vs. control groups (3.11% vs. 2.03%, P<0.01), (b) the frequency of SCE per cell (6.96 vs. 4.87, P<0.001), and (c) the percentage of HFC (19.9% vs. 4.1%, P<0.001). BD exposure had no significant effects on formation of micronuclei and on comet assay parameters. Effect of smoking was observed only for HFC in BD-exposed group. GSTM1 genotype affected chromosomal aberrations in exposed group, while GSTT1 genotype affected chromosomal aberrations in controls. No effect of GSTM1 or GSTT1 genotypes was observed on any other biomarkers used.     

Serum antibodies reacting with subgingival species in refractory periodontitis subjects

The purpose of this investigation was to compare the levels of serum IgG antibody to 85 subgingival species in 32 refractory periodontitis, 56 successfully treated, and 33 periodontally healthy subjects. Refractory subjects showed mean full mouth attachment loss and/or >3 sites showing attachment loss >2.5 mm within 1 year after 2 treatment modalities, scaling and root planing and surgery plus systemically administered tetracycline. Successfully-treated subjects showed mean attachment level gain and no sites with attachment loss >2.5 mm, 1 year post-therapy. Periodontally healthy subjects exhibited no pocket or attachment level >3 mm, and no evidence of progressing attachment loss during 1 year of monitoring. Baseline serum was obtained from each subject and tested against 85 subgingival species, including reference strains and strains isolated from refractory subjects, using checkerboard immunoblotting. Significance of differences in levels of serum antibody among groups were sought using the Kruskal-Wallis test. Refractory subjects constituted a heterogeneous group based on their serum antibody response to subgingival species. Some individuals had antibody reactions to many subgingival species, while other subjects showed fewer or low numbers of responses. On average, refractory subjects exhibited higher numbers and levels of serum antibody reactions to a wide range of subgingival species than successfully treated or periodontally healthy subjects. Differences in serum antibody among clinical groups were more striking at higher threshold levels of antibody (>50 microg/ml and > 100 microg/ml). The data showed that a subject was 10.1 x more likely to be refractory if the subject exhibited antibody reactions with >9 subgingival species at >50 microg/ml (p<0.001, after adjusting for multiple comparisons). Serum antibody to a subset of the test species differed among the clinical groups. Porphyromonas gingivalis, Bacteroidesforsythus, and some strains isolated from refractory subjects (a novel Neisseria sp., Enterococcus faecalis, Prevotella loescheii and Prevotella oulora) elicited high serum antibody in the successfully treated and refractory subjects. High levels of serum antibody to a Microbacterium lacticum-like organism, Streptococcus oralis, Streptococcus constellatus, Actinobacillus actinonmycetemcomitans serotype c and Haemophilus aphrophilus significantly increased the likelihood of a subject being refractory to conventional periodontal therapy.