The human whole saliva proteome was investigated using two-dimensional liquid chromatography (2-DLC). The 2-DLC study was able to identify, with high confidence, 102 proteins including most known salivary proteins (35), and a large number of common serum proteins (67). Peptides from proline-rich proteins, abundant in saliva, had unusual cleavage sites and were frequently only partially tryptic. Three proteins not previously observed in human saliva were also detected. Significantly greater numbers of identified proteins, including high molecular weight, low molecular weight, and proline-rich proteins, were found with 2-DLC compared to previously reported two-dimensional gel electrophoresis studies.

A novel mouthrinse (IND 61,164) containing essential oils and extracts from four plant species (Melaleuca alternifolia, Leptospermum scoparium, Calendula officinalis and Camellia sinensis) were tested. This study aimed to evaluate the safety, palatability and preliminary efficacy of the rinse. Fifteen subjects completed the Phase I safety study. Seventeen subjects completed the Phase II randomized placebo-controlled study. Plaque was collected, gingival and plaque indices were recorded (baseline, 6 weeks, and 12 weeks). The relative abundance of two periodontal pathogens (Actinobacillus actinomycetemcomitans, Tanerella forsythensis) was determined utilizing digoxigenin-labeled DNA probes. ANCOVA was used at the p = 0.05 level of significance. Two subjects reported a minor adverse event. One subject withdrew from the study. Several subjects objected to the taste of the test rinse but continued treatment. Differences between gingival index, plaque index or relative abundance of either bacterial species did not reach statistical significance when comparing nine placebo subjects with eight test rinse subjects. Subjects exposed to the test rinse experienced no abnormal oral lesions, altered vital signs, changes in liver, kidney, or bone marrow function. Larger scale studies would be necessary to determine the efficacy and oral health benefits of the test rinse.

The time course of ICAM-1 expression and leukocyte subset infiltration was studied in a model of CNS reperfusion injury in adult rats. Leukocyte adhesion and infiltration, mediated in part by intercellular adhesion molecule-1 (ICAM-1), appears to potentiate CNS reperfusion injury. The timing and relationship between ICAM-1 staining and leukocyte infiltration postglobal CNS ischemia is unknown. Reversible forebrain ischemia was produced in 32 adult Sprague-Dawley rats using the two-vessel occlusion model with histologic analysis performed at specific intervals postischemia: 1, 3, 6, 12, and 24 h, 4 and 7 d, or sham-operated controls (n = 4 each group). Monoclonal antibodies against ICAM-1 (1A29 and TM8), a specific granulocyte (PMN) (HIS48), and a specific monocyte/macrophage (M phi) (ED1) were used. No specific leukocyte and only rare ICAM-1 vessel immunoreactivity was observed in sham controls. ICAM-1: Significant expression in microvessels beginning at 1 h with additional diffuse CA1 pyramidal layer staining beginning at 4 d. Leukocytes: No PMN cells and rare M phi identified at 6 and 12 h. By 24 h: moderate infiltrate in areas of ICAM-1 expression of PMN and M phi. At 4 and 7 d: only M phi accumulation, cellular morphology now similar to microglia. The results of this study indicate that early and persistent ICAM-1 expression occurs following CNS ischemia with associated leukocyte infiltration.

The mechanisms mediating cAMP effects to stimulate transcription of the PRL gene have been examined. Treatments that elevate intracellular cAMP concentrations were found to stimulate the mitogen-activated protein kinase (MAPK) in GH(3) cells. Elevated cAMP was also found to stimulate activation of the GTP-binding protein, Rap1. Rap1GAP1 reduced cAMP-induced phosphorylation of MAPK, offering evidence that Rap1 may play a role in mediating activation of MAPK. Treatment of GH(3) cells with PD98059, an inhibitor of the MAPK pathway, reduced the ability of forskolin to activate a PRL reporter gene, providing evidence that MAPK contributes to cAMP-mediated effects on the PRL promoter. As previous studies have implicated Ets factor binding sites within the PRL promoter in mediating responses to MAPK, we expected that the Ets sites would also play a role in cAMP responsiveness. Surprisingly, mutation of all of the consensus Ets factor binding sites in the proximal PRL promoter greatly reduced responsiveness to epidermal growth factor (EGF) and TRH but did not reduce cAMP responsiveness. Experiments using an expression vector for adenovirus 12S E1a provided evidence that the coactivators, CREB binding protein and/or p300, probably play a role in cAMP responsiveness of the PRL promoter. Interestingly, the ability of a GAL4-p300 fusion protein to enhance reporter gene activity was stimulated by cAMP in a MAPK-dependent manner. These findings provide evidence for a model for cAMP-induced PRL transcription involving Rap1-induced MAPK activity leading to stimulation of the transcriptional coactivators, CBP and p300.

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