RIP1 links inflammatory and growth factor signaling pathways by regulating expression of the EGFR.

There is considerable interest in understanding how inflammatory responses influence cell proliferation and cancer. In this study, we show that the receptor-interacting protein (RIP1), a critical mediator of inflammation and stress-induced NF-kappaB activation, regulates the expression of the epidermal growth factor receptor (EGFR). Mouse embryo fibroblasts (MEFs) derived from RIP1 knockout mice express very high levels of the EGFR. Reconstitution of RIP1(-/-) MEFs with RIP1 results in a lowering of EGFR levels. RIP1 influences EGFR at the mRNA level by regulating the EGFR promoter. Expression of RIP1 inhibits the EGFR promoter. RIP1 downregulates EGFR expression by interfering with the function of Sp1, which is a key activator of EGFR transcription. RIP1 suppresses Sp1 activity and overexpression of Sp1 reverses RIP1-mediated repression of the EGFR promoter. RIP1 is present both in the cytoplasm and in the nucleus. RIP1 coimmunoprecipitates with Sp1 in vivo and binds directly to Sp1 in vitro. A RIP1 mutant lacking the death domain fails to suppress Sp1 activity and the EGFR promoter, suggesting a critical role for the RIP1 death domain in EGFR regulation. Thus, our study identifies a new link between inflammatory and growth factor signaling pathways mediated by RIP1 and provides insight into the mechanism used by RIP1 to regulate EGFR levels.

Two implants for all edentulous mandibles.

Complete dentures have always been a poor substitute for natural teeth. Mandibular complete dentures frequently cause pain and discomfort, accelerated residual bone resorption, while failing to restore effective chewing. The provision of two implants to stabilise the mandibular complete denture can result in significant improvements.

Oral health impact on daily performance in patients with implant-stabilized overdentures and patients with conventional complete dentures.

This cohort study (n = 83) investigated whether patients with implant-stabilized overdentures would demonstrate less impact on daily life, would have less difficulty in the mastication of different types of food, and would generally be more satisfied than patients with conventional complete dentures. The groups were comparable for gender, age of dentures, and duration of edentulism. The patients were interviewed using a questionnaire, which included the Oral Impacts on Daily Performances (OIDP) sociodental indicator. Patients with implant-stabilized overdentures were more satisfied with the comfort of their dentures, could eat a wide range of food items with less difficulty, and experienced less impact on daily life than patients with conventional complete dentures. The findings of this study support the need to consider implant-stabilized overdentures in the treatment of edentulous patients.

The design and synthesis of purine inhibitors of CDK2. III.

Cyclin-dependent kinases (CDKs) belong to a class of enzymes that control the ability of a cell to enter into and proceed through the cell division cycle. Using purine as a scaffold, we have synthesized a number of nanomolar inhibitors of CDK-2/cyclin E. In this report, the synthesis of a series of piperidine-substituted purine analogs will be presented, as well as some of their in vitro and in vivo biological effects.

Expression of p21Waf1/Cip1 and cyclin D1 is increased in butyrate-resistant HeLa cells.

Sodium butyrate induced cell cycle arrest in mammalian cells through an increase in p21Waf1/Cip1, although another study showed that this arrest is related to pRB signaling. We isolated variants of HeLa cells adapted to growth in 5 mm butyrate. One of these variants, clone 5.1, constitutively expressed elevated levels of p21Waf1/Cip1 when incubated in regular growth medium and in the presence of butyrate. Despite this elevated level of p21Waf1/Cip1, the cells continue to proliferate, albeit at a slower rate than parental HeLa cells. Western blot analyses showed that other cell cycle regulatory proteins were not up-regulated to compensate for the elevated expression of p21Waf1/Cip1. However, cyclin D1 was down-regulated by butyrate in HeLa cells but not in clone 5.1. We conclude that continued expression of cyclin D1 allowed clone 5.1 to grow in the presence of butyrate and elevated levels of p21Waf1/Cip1.

Crystal structure of human cyclin-dependent kinase 2 in complex with the adenine-derived inhibitor H717.

Cyclin-dependent kinases (CDKs) are regulatory proteins of the eukaryotic cell cycle. They act after association with different cyclins, the concentrations of which vary throughout the progression of the cell cycle. As central mediators of cell growth, CDKs are potential targets for inhibitory molecules that would allow disruption of the cell cycle in order to evoke an antiproliferative effect and may therefore be useful as cancer therapeutics. We synthesized several inhibitory 2,6,9-trisubstituted purine derivatives and solved the crystal structure of one of these compounds, H717, in complex with human CDK2 at 2.6 A resolution. The orientation of the C2-p-diaminocyclohexyl portion of the inhibitor is strikingly different from those of similar moieties in other related inhibitor complexes. The N9-cyclopentyl ring fully occupies a space in the enzyme which is otherwise empty, while the C6-N-aminobenzyl substituent points out of the ATP-binding site. The structure provides a basis for the further development of more potent inhibitory drugs.

Effects of attachment type on the mobility of implant-stabilized overdentures--an in vitro study.

PURPOSE: To stabilize overdentures, a wide range of attachments to implants is suggested. Although there is evidence that denture stability is an important factor for patient satisfaction, there are no data on how these attachments may reduce denture mobility. It was the purpose of this study to compare the effects of different types of attachments on the mobility of implant-stabilized overdentures in vitro, designing a measurement device that could also be used in vivo. MATERIALS AND METHODS: On an acrylic model with 2 implants in the canine areas, magnets were fixed to one of the implant abutments. Four Hall-effect devices were attached to the denture opposite the magnet, which allowed contact-free measurements of denture movements. RESULTS: In vitro experiments loading an overdenture showed very small, largely insignificant differences in denture mobility when different bar or ball attachments were used. Geometric aspects of load application were more important than the choice of attachment. CONCLUSION: The measurements gave no guide to the choice of an attachment. The similarity of the attachments must be confirmed by in vivo measurements.

Simplified method of estimating masticatory performance.

Methods which measure masticatory performance include gravimetric, volumetric and direct observation which depend on the weight, volume and size, respectively, of a test food once chewing is completed. Almonds, one of the most common test foods used, have a convenient size and texture. However, due to their oily content and mixing with saliva, washing and drying is required to overcome the clumping of chewed particles. A method has been developed using bagged almonds to exclude saliva and preventing loss of almond particles in the mouth. In addition, microwaving the whole almond reduces the oil content of the almond, reducing clumping, and potentially eliminating the need for washing and drying the particles. A dentate volunteer was asked to chew seven blanched almonds and seven microwaved almonds. The chewed particles were separated using two sieves, weighed and optically scanned to measure the number and area of the particles. Results were obtained both before and after washing and drying of the chewed particles. The overall results for the test of masticatory performance is very similar whether or not a washing stage is used for microwaved almonds. For untreated almonds washing has a more noticeable effect and may still be considered necessary.

Induction of vascular cell adhesion molecule-1 expression by IL-4 in human aortic smooth muscle cells is not associated with increased nuclear NF-kappaB levels.

Vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) are upregulated in vascular endothelial and smooth muscle cells by cytokines produced at sites of inflammation. The cytokine profile for induction of VCAM-1, however, is different for the two cell types. Tumor necrosis factor-alpha (TNF-alpha) induced both VCAM-1 and ICAM-1 expression in human umbilical vein endothelial cells (HUVECs; ED50 approximately 300 and 30 U/ml, respectively). TNF-alpha and interleukin-1beta (IL-1beta) stimulated cell surface ICAM-1 expression, but not VCAM-1 expression, in human aortic smooth muscle cells (HASMCs). Conversely, IL-4 was a potent VCAM-1 inducer in HASMCs (ED50 approximately 100 pg/ml) but did not induce ICAM-1 expression. Nuclear extracts from IL-4-treated cells were compared with untreated cells for relative nuclear factor-kappa B (NF-kappaB) levels by using an electrophoretic mobility shift assay and surface plasmon resonance techniques. No significant increase in nuclear NF-kappaB DNA binding activity was detected in IL-4-treated HASMCs by either method of analysis. IL-1beta and TNF-alpha stimulated nuclear NF-kappaB levels by about fourfold and fivefold, respectively, in HASMCs. The antioxidant pyrrolidine dithiocarbamate (PDTC) similarly inhibited VCAM-1 upregulation in HASMCs incubated with IL-4 and in HUVECs incubated with TNF-alpha (IC50s of 25 and 40 microM, respectively). These data suggest that a significant increase in nuclear NF-kappaB levels is not necessary or sufficient for VCAM-1 upregulation in HASMCs and does not determine the relative sensitivity to inhibition of gene expression by PDTC.

Microautoradiographic quantitation of vascular endothelial growth factor mRNA levels in human prostate specimens containing normal and neoplastic epithelium.

Human prostate specimens commonly contain a spectrum of epithelial changes, including normal acinar and ductal structures, hyperplasia, intraepithelial neoplasia (dysplasia), and carcinoma. Since vascular endothelial growth factor (VEGF) expression is dependent on cell type and tissue microenvironment, meaningful quantitation of the levels of this mRNA in pathological specimens requires analysis at the microscopic level. Phosphorimage analysis of the binding of radiolabeled cRNA probes to tissue sections allows quantitation of mRNA levels, but the resolution is limited. Alternatively, emulsion autoradiography allows visualization of mRNA levels at cellular resolution, but quantitation is difficult. We have developed a method of quantitating steady state mRNA levels in tissue sections at the microscopic level, using autoradiography and quantitative image analysis. In this study, we describe the method and apply it to quantitation of VEGF mRNA in human prostate specimens. The VEGF mRNA level was low in nonepithelial stromal tissue (0.8 dpm/mm2), high in normal and benign hyperplastic epithelium (17-18 dpm/mm2), and significantly decreased in intraepithelial neoplasia (6.4 dpm/mm2) and in microacinar carcinoma that had invaded the stroma (3.5 dpm/mm2). Immunohistochemical staining detected VEGF protein in epithelial and stromal cells, with highest levels on the luminal surface of normal epithelium and in stromal cells, and lower levels in benign hyperplasia, intraepithelial neoplasia, and carcinoma. No correlation between VEGF expression in epithelium and nearby vessel density was observed. The results indicate a decrease in the steady state level of VEGF mRNA when prostate epithelial cells become transformed, escape the confines of glandular structure and invade the stroma, and suggest that the progression of prostatic carcinoma through the stages examined in this study is not associated with increased VEGF expression, in contrast to the elevated VEGF expression associated with progression of several other tumor types.

Clomiphene analogs with activity in vitro and in vivo against human breast cancer cells.

Six hundred triphenylethylenes were assayed for antiproliferative activity against MCF-7, LY2, and MDA-MB-231 breast cancer cells using sulforhodamine B dye to measure proliferation. Here we report on just 63 of the compounds, mostly clomiphene analogs, with substitutions on the alpha' or beta ring, at the vinyl position or in the side chain, of which 23 were active, as defined by antiproliferation IC50 values < or =1 microM. Activity profiles showed that 23 and 11 analogs were active toward MCF-7 and LY2, respectively, but none were active against MDA-MB-231. The IC50 values of tamoxifen were 2.0 microM against MCF-7 and 7.5 microM against LY2 and MDA-MB-231. Estradiol reversed antiproliferative activities of several E isomers but not their Z isomer counterparts. Clomiphene side chain analogs 46 [(E)-1-butanamine, 4-[4-(2-chloro-1,2-diphenylethenyl) phenoxy]-N,N-diethyl-dihydrogen citrate (MDL 103,323)] and 57 [(E)-N-[p-(2-chloro-1,2-diphenylvinyl) phenyl]-N,N-diethylethylenediamine dihydrogen citrate (MDL 101,986)] were 4- to 5-fold more effective than tamoxifen. Methylene additions up to (-CH2-)12 in the clomiphene side chain showed that analog 46 [(-CH2-)4 side chain] had maximal antiproliferative activity, binding affinity, and inhibition of transcription of an estrogen response element luciferase construct in transfected MCF-7 cells. Intraperitoneal administration of 46 or 57 inhibited progression of MCF-7 breast tumor xenografts in nude mice with ED50 values of <0.02 mg/mouse/day. Both analogs may hold promise for treating ER positive breast cancer and are of interest for further development.

Evaluating the effect of soft lining materials on the growth of yeast.

STATEMENT OF PROBLEM: Soft lining materials continue to have a place in clinical removable prosthodontics. However, there is an increased probability of yeast colonization on soft lining materials. PURPOSE: This study (1) assessed a method of evaluating the effect of long-term soft lining materials on the growth of yeast and (2) investigated the effect five soft lining materials had on the growth of three species of yeast. MATERIAL AND METHODS: Coe Supersoft, Novus, and three experimental soft lining materials were investigated together with Candida albicans, Candida tropicalis, and Issatchenkia orientalis (formerly Candida krusei) yeasts. Strips of soft lining material incubated on blood agar plates were examined for inhibition of the growth of yeast. Soft lining materials soaked in sterile trypticase soya broth or water were inoculated with yeast and incubated. The change in colony forming units per milliliter from the initial load of yeast at 3 days was measured. Statistical analysis was performed with an independent paired Student t test. RESULTS: Inhibition of yeast growth occurred for two soft lining materials. Despite the presence of sufficient viable organisms, differences between the initial load of yeast and the 3-day results were mostly small, both for the test and control groups, suggesting that the material does not support the growth of the tested yeast during this period. CONCLUSIONS: The often described increased prevalence of yeast associated with soft lining materials in the oral environment is likely related to readily available nutrients in the mouth and the difficulty in maintaining and cleaning these materials.

Use of microwave energy to disinfect a long-term soft lining material contaminated with Candida albicans or Staphylococcus aureus.

STATEMENT OF PROBLEM: Soft lining materials have been found to be more susceptible to microbial adhesion than acrylic resin base materials. Denture hygiene is essential to maintain the serviceability of the denture, and microwave energy has been suggested for denture disinfection. PURPOSE: The purpose of this study was to determine the effectiveness of microwave energy in the disinfection of a long-term soft lining material. MATERIAL AND METHODS: A long-term soft lining material was contaminated with known microorganisms and the reduction of organism counts after test disinfection regimes calculated. The disinfection regimes were microwaving for 5 minutes, leaving dry overnight, and soaking overnight in a dilute sodium hypochlorite solution. The test microorganisms were Candida albicans or Staphylococcus aureus. RESULTS: For both organisms, soaking in sodium hypochlorite reduced the number of viable adherent microorganisms recovered significantly more than exposure to microwave energy, which led to greater reduction than leaving the lining material dry overnight (p < 0.001, Wilcoxon nonparametric signed rank test). CONCLUSION: With reference to the tested microorganisms, disinfection of Molloplast-b soft lining material in dilute sodium hypochlorite solution proved to be more effective than exposure to microwave energy, which in turn was more effective than leaving the lining dry overnight.

Effect of prefabricated bar design with implant-stabilized prostheses on ridge resorption: a clinical report.

This investigation was concerned with the resorption of the posterior mandibular residual ridge in patients wearing mandibular overdentures supported by either parallel-sided bars (rigid joint) or oval straight bars (resilient joint) on two activated implants. Rotational tomographs were taken shortly after implant placement and up to 8 years later. Using proportional measurement, the area of residual ridge was measured in bilateral posterior areas. Patients rehabilitated with implant-stabilized mandibular overdentures demonstrated posterior mandibular residual ridge resorption at low rates, which were not significantly influenced by the design of the prefabricated bar.

Adhesion and tear energy of a long-term soft lining material activated by rapid microwave energy.

STATEMENT OF PROBLEM: Construction of dentures with permanent soft linings is time-consuming in the laboratory and extra costs are related to equipment and materials used. PURPOSE: The purpose of this in vitro study was to determine whether using microwave energy to activate the polymerization of a silicone rubber denture soft lining material affected its properties. MATERIAL AND METHODS: Tear energy and adhesive properties were measured in a tensile testing machine by using a pants leg tear test and peel specimens. Tear energy was measured for specimens polymerized conventionally (control) and for 3, 5, and 10 minutes in a microwave. Data were analyzed with one-way analysis of variance and a two-sample Student t test. RESULTS: The multiple comparison test failed to show a significant difference in tear energy between 3 minutes microwave activation and conventional heat curing. However, 3 minutes microwave activation revealed a significantly stronger material when compared with 5 minutes and 10 minutes (p < 0.05). Application of a two-sample Student t test failed to demonstrate a significant difference between microwave energy and conventional heat activation groups in the adhesion test. In adhesion testing, all specimens presented cohesive failure. CONCLUSIONS: This method of polymerization does not compromise the strength of a soft lining material and its adhesion to polymethyl methacrylate. This study suggests the use of 3 minutes 650 W microwave energy for processing a silicone soft lining material.

Chewing performance and occlusal contact area with the shortened dental arch.

The aim of this study was to investigate the effect of the loss of posterior teeth on the effectiveness of mastication. To evaluate this, chewing performance and occlusal contact area were investigated in 10 edentate subjects having implants stabilising a mandibular overdenture. A copy of the original lower denture was made for each subject, with removable teeth, which could be separated to convert a complete dental arch to a shortened dental arch, an extremely shortened dental arch, or a broken dental arch. Both post canine occlusal contact area and chewing performance demonstrated significant differences between the different arches. It was concluded that chewing performance is reduced by removing posterior teeth from implant stabilised mandibular complete dentures.

Cyclic AMP regulation of mouse proline-rich protein gene expression: isoproterenol induction of AP-1 transcription factors in parotid glands.

Proline-rich protein mRNAs are increased dramatically in the salivary glands of rats, mice, and hamsters upon treatment with the beta-agonist isoproterenol. Sequence comparisons between mice and hamster proline-rich protein genes identified conserved regions upstream from the transcription start site. Reporter plasmids containing these 5'-flanking sequences from a mouse proline-rich protein gene, MP2, were constructed and tested for transcriptional regulation by cAMP. Transient transfection experiments in mouse L-M cells showed that the upstream region -702 to -322 bp relative to the transcription start site is sufficient to confer cAMP induction on a heterologous promoter. Multiple copies of the AP-1 sequence elements within this region (-625 to -551) mediate the cAMP transcriptional response of reporter gene expression in L-M cells. L-M cell nuclear proteins and purified human c-jun protein bind to these upstream elements as determined by DNase I footprint analysis. Nuclear proteins isolated from mouse parotid glands protected the consensus AP-1 binding site 5'-TGAGTCA-3' (-592 to -586). The nuclear proteins interacting at this site were increased about sixfold in glands isolated from isoproterenol-treated mice when compared with glands from untreated mice. These results suggest that induction of AP-1 transcription factors in the parotid gland control the upregulation of some mouse salivary proline-rich proteins.

Quantitation of vascular endothelial growth factor mRNA levels in human breast tumors and metastatic lymph nodes.

In situ hybridization analysis provides a means to qualitatively study the heterogeneity of primary tumors and metastases based on the types of genes transcribed. In this study, we have tested some parameters for quantitative analysis of in situ hybridizations with paraffin-embedded human breast tumors and measured mRNA levels for the angiogenic protein, vascular endothelial growth factor (VEGF). VEGF mRNAs were highly tumor specific, with the highest levels near necrotic regions within the tissues (0.1 to 2.7 dpm/mm2). Normal cells within the tissue sections did not have detectable levels of VEGF mRNA. For comparison, tumor levels of c-myc (4 to 46 dpm/mm2) and glyceraldehyde-3-phosphate dehydrogenase mRNAs (48 to 214 dpm/mm2) were measured. The mRNAs for both of these genes were more broadly expressed across the tissue sections. The hybridization pattern for VEGF mRNAs was consistent with hypoxia-induced VEGF mRNA steady-state levels and supports the hypothesis that oxidative stress regulates VEGF expression in breast tumors.