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Selectivity, efficiency, and surface characteristics of hard dental tissues ablated with ArF pulsed excimer lasers.

Lasers are finding expanding applications in the field of dentistry. Cutting in soft tissue, hard dental material ablation, caries removal, and root canal therapy are only a few examples of dental laser uses. In this article, the application of short pulse ArF excimer laser to ablation of dentin and enamel is investigated. In particular, the effect of laser pulse repetition rates (PRR) and fluence levels on the efficiency of the ablation process and on the average thermal response of ablated surfaces is investigated. Ablation of dentin was found to be considerably more efficient than the ablation of enamel and depends exponentially on the laser fluence. Both dentin and enamel surfaces showed an increase in surface temperature with repetition rate. At lower PRR, however, temperature increases are very small. Surface temperature was also found to increase with laser fluence, although this increase is very small at laser PRR of 5 Hz or less. Tissue ablation rates were found to be comparable to or better than other nanosecond lasers, and left smooth surfaces, free of thermal damage. Microscopic examination of the ablated surface shows no crack formation, charring, discoloration, or any other thermal damage. The ablated surfaces appear to be very smooth, highly polished, and glossy looking as if they were subjected to thermal melting. This observation is indeed confirmed under scanning electron microscopy (SEM), where evidence of localized melting of the tissue is observed. Furthermore, a close SEM examination of the dentin surface reveals a selectively ablated intertubular dentin, while the remaining pillar-like dentin tubules are sealed off with fused peritubular dentin. At all fluence levels and PRR, the first three to four pulses impinging on an untreated enamel surface produced unusually large plumes of debris which were different in size, texture, and fluorescence emission characteristics from the ablation products of subsequent pulses. It is believed that these different ablation characteristics are a consequence of the pulsed ArF laser's ability to selectively remove residual matter from the more resistant enamel surface.

Acid Etching, Dental↗

Effect of the ArF excimer laser on human enamel.

Human enamel surface was irradiated with ArF excimer laser and examined under light microscopy and scanning electron microscopy (SEM). Enamel surface was irradiated at three different areas with different energy fluences. It is demonstrated that the ArF excimer laser causes ablation of the calcified hard enamel tissue. Ablation curves were measured. There was no significant difference found in the etch depth between the three different areas of enamel surface. The morphology of the irradiated areas seen under the SEM was found to be dependent on energy fluence. It changed with increase in energy fluence from being etched to forming a smooth, fused, glaze-like surface and then at very high energy fluences producing a rough surface. The influence of the laser irradiation was confined to the irradiated area only, with no visible heat damage to the surroundings. These results suggest that excimer laser could be applied in a controlled and defined manner for tooth enamel treatments in dentistry.

Dental Enamel↗

ArF 193 nm excimer laser corneal surgery as a possible risk factor in cataractogenesis.

It was evaluated whether ArF 193 nm excimer laser corneal surgery represents a risk factor in cataractogenesis. For this purpose, 20 male albino rabbits underwent a photorefractive keratectomy and biochemical analyses on aqueous humour (hydrogen peroxide, ascorbic acid, and reduced and oxidized glutathione) and on lens (malondialdehyde, reduced and oxidized glutathione) were performed. In the aqueous humour of all treated animals a significant increase in hydrogen peroxide and in oxidized glutathione and a concomitant decrease in ascorbic acid and reduced glutathione concentration were observed. Moreover, all these variations were significantly correlated with the cumulative UV dose used. In the lens, after excimer laser corneal surgery, there was a dramatic loss of reduced glutathione and a parallel increase in oxidized glutathione levels. Malondialdehyde concentration was also increased, but only at the highest UV exposure. Moreover, all these variations were significantly correlated with the cumulative UV dose used. These findings demonstrate that the exposure of aqueous humour and lens to the secondary radiation generated after ArF 193 nm excimer laser corneal photoablative keratectomy induces biochemical modifications which are known to be markers of cataractogenesis.

Animals↗

3-Morpholinosydnonimine hydrochloride induces p53-dependent apoptosis in murine primary neural cells: a critical role for p21(ras)-MAPK-p19(ARF) pathway.

In some neurological disorders, excessive nitric oxide (NO, nitrogen monoxide) produced by inducible and/or neuronal nitric oxide synthases (iNOS and nNOS) is able to combine with superoxide (O(minus sign)(2)) to form peroxynitrite (ONOO(minus sign)), which can then induce p53-dependent neural apoptosis. In the present study, experiments using p53 knock-out mice primary neural cells revealed that 3-morpholinosydnonimine hydrochloride (SIN-1), a peroxynitrite donor, triggered apoptosis, while p53-transcriptional activity was effectively suppressed in the absence of p53 molecules. This shows that SIN-1 was able to induce p53-dependent apoptosis in murine primary neural cells. The mechanism responsible for the SIN-1-induced accumulation of p53 molecules was then analyzed. Western blot analysis indicated that p53 accumulation caused by SIN-1 did not require p53 phosphorylation, whereas SIN-1 treatment triggered MAP kinase (MAPK) phosphorylation and pretreatment with the MAP kinase kinase (MEK) inhibitor U0126 inhibited p53 accumulation. Pretreatment of the neural cells with lovastatin, an inhibitor of p21(ras) signaling, greatly inhibited the accumulation of p53 induced by SIN-1. Northern blot and immunofluorescence analyses revealed that primary neural cells treated with SIN-1 had increased levels of p19 alternate reading frame (p19(ARF)) mRNA and protein, which is induced by MAPK and stabilizes the p53 protein. Our findings clearly show that the p21(ras)-MAPK-p19(ARF) pathway has an essential role in p53-dependent apoptosis triggered by peroxynitrite in neural cells.

Animals↗

A Zea mays GTP-binding protein of the ARF family complements an Escherichia coli mutant with a temperature-sensitive malonyl-coenzyme A:acyl carrier protein transacylase.

In an attempt to isolate a plant malonyl-coenzyme A:acyl carrier protein transacylase cDNA clone, by direct genetic selection in an Escherichia coli fabD mutant (LA2-89) with a maize cDNA expression library, a Zea mays cDNA clone encoding a GTP-binding protein of the ARF family was isolated. Complementation of a mutation affecting bacterial membrane lipid biosynthesis by a plant ARF protein, could indicate the existence of as yet unidentified bacterial equivalents of this ubiquitous eucaryotic GTP-binding protein.

ADP-Ribosylation Factors↗

Controlled ventilation with best positive end-expiratory pressure (PEEP) and high level PEEP in acute respiratory failure (ARF).

We assessed hemodynamics, total lung and chest wall compliance (CT) and gas exchange using two different levels of PEEP during controlled ventilation in two different groups of patients with ARF; in the first group (Group 1, 12 patients) chest X-Rays showed a symmetrical pattern of bilateral alveolar infiltrates; in the second group (Group 2, 5 patients) chest X-Ray showed a asymmetrical pattern with unilateral lobar consolidation. A first level of PEEP (best PEEP = 9 +/- 3 cm H2O) produced an improvement in CT and in gas exchange with a slight decrease in cardiac index in both groups; but improvement in PaO2 (from 64 +/-33 to 122 +/- 76 torr, p less than 0.001 in Group 1, and from 76 +/- 39 to 91 +/- 33 torr, p less than 0.05 in Group 2) and decrease in QS/QT were not as well marked in Group 2 as i Group 1. A second level of PEEP (high level PEEP: 20 +/- 4 cm H2O) produced a sharp decrease in CT and required hemodynamic support in each case (blood volume expansion with or without Dopamine infusion) to maintain cardiac index within a normal range. In Group 1 this high level PEEP produced a greater improvement in gas exchange (PaO2 increased from 122 +/- 76 to 194 +/- 76, p less than 0.01) but in Group 2 it had a deleterious effect, producing a decrease in PaO2 (from 91 +/- 33 to 76 +/- 41 torr, p less than 0.05), and an increase in QS/QT; with this higher PEEP we also noted an increase of alveolar dead space in Group 2. This study demonstrates the efficiency of high levels of PEEP to reduce QS/QT in ARF but also shows its limitations: namely reduction in cardiac performance and in efficiency if the damage to one lung is significantly more pronounced than that to the other lung.

Acute Disease↗

ARF-regulated phospholipase D: a potential role in membrane traffic.

Phospholipase D activity is stimulated rapidly upon occupation of cell-surface receptors. One of the intracellular regulators of phospholipase D activity has been identified as ADP ribosylation factor (ARF). ARF is a small GTP binding protein whose function has been elucidated in vesicular traffic. This review puts into context the connection between the two fields of signal transduction and vesicular transport.

ADP-Ribosylation Factors↗

Coupling actin and membrane dynamics during calcium-regulated exocytosis: a role for Rho and ARF GTPases.

Release of neurotransmitters and hormones occurs by calcium-regulated exocytosis, a process that shares many similarities in neurons and neuroendocrine cells. Exocytosis is confined to specific regions in the plasma membrane, where actin remodelling, lipid modifications and protein-protein interactions take place to mediate vesicle/granule docking, priming and fusion. The spatial and temporal coordination of the various players to form a "fast and furious" machinery for secretion remain poorly understood. ARF and Rho GTPases play a central role in coupling actin dynamics to membrane trafficking events in eukaryotic cells. Here, we review the role of Rho and ARF GTPases in supplying actin and lipid structures required for synaptic vesicle and secretory granule exocytosis. Their possible functional interplay may provide the molecular cues for efficient and localized exocytotic fusion.

ADP-Ribosylation Factors↗

The Arf-family protein, Arl8b, is involved in the spatial distribution of lysosomes.

Lysosomes are late-endocytic organelles which primarily contribute to degradation and recycling of cellular material. From a previous proteomics study of purified rat liver lysosomal membranes we identified a protein from the Arf-family of small GTPases, Arl8b. Although proteins of the Arf-family have roles in a wide range of cellular functions, most notably roles in protein/vesicular trafficking, Arl8b represents the first from this protein family to be associated with a late-endocytic organelle. We demonstrate the co-localization of this protein with various lysosomal markers in different cell lines by confocal-immunofluorescence microscopy. We also show that GTP-restricted mutant Arl8b localizes to lysosomes and causes their redistribution to the periphery of the cell and into membrane projections. This indicates that Arl8b is involved in trafficking processes for lysosomes.

ADP-Ribosylation Factors↗

Antagonism of Myc functions by Arf.

The Arf-Mdm2-p53 tumor suppressor pathway is activated by sustained hyperproliferative signals emanating from oncoproteins such as Myc. A recent study reveals a novel level of feedback control, whereby induced p19(Arf) binds to Myc and blocks cell proliferation by selectively impairing its transactivation functions.

Animals↗

The co-transfection of p16(INK4a) and p14(ARF) genes into human lung cancer cell line A549 and the effects on cell growth and chemosensitivity.

Two functionally and structurally different proteins, p16(INK4a) and p14(ARF), encoded by the gene INK4a/ARF located at 9p21 are cyclin-dependent kinase (cdk) inhibitors and important cell cycle regulators. More and more evidences have been accumulated to show that the exogenous p16(INK4a) or p14(ARF) can inhibit the cell growth and/or induce the apoptosis. But it is still unclear if they can play positive role when combine with the conventional chemotherapy in cancer treatment. Here we show that cationic liposome-mediated gene transfection of INK4a/ARF into lung cancer cell line A549, in which the INK4a/ARF locus was lost, suppressed the growth and induced apoptosis. When treated with five different chemotherapy drugs with different mechanism after the transfection, A549 got an increased chemosensitivity for adriamycin and cisplatin and an unchanged result for topotecan, taxol or vinorelbine. The results indicated that cell cycle redistribution and increased apoptosis index after transfection might be the main explanation for the enhanced chemosensitivity. The combination of gene therapy with conventional chemotherapy is not always better than single chemotherapy. This trial will be of benefit to the treatment of lung cancer when combine the conventional chemotherapy and gene therapy in the future.

Antineoplastic Agents↗

ADP-ribosylation factor (ARF)-like 7 (ARL7) is induced by cholesterol loading and participates in apolipoprotein AI-dependent cholesterol export.

Here, we identify ADP-ribosylation factor (ARF)-like 7 (ARL7) as the only ARF- and ARL-family member whose mRNA-expression is induced by liver X-receptor/retinoid X-receptor agonists or cholesterol loading in human macrophages. Moreover, subcellular distribution of mutant and wild type ARL7-enhanced green fluorescent protein (EGFP) supports that ARL7 may be involved in a vesicular transport step between a perinuclear compartment and the plasma membrane. Therefore, we investigated the effect of ARL7 over-expression on the cholesterol secretory pathway. We found that expression of wild type and dominant active ARL7-EGFP stimulated the rate of apolipoprotein AI-specific cholesterol efflux 1.7- and 2.8-fold. In contrast, expression of the dominant negative form of ARL7-EGFP led to approximately 50% inhibition of cholesterol efflux. This data is consistent with a model in which ARL7 is involved in transport between a perinuclear compartment and the plasma membrane apparently linked to the ABCA1-mediated cholesterol secretion pathway.

ADP-Ribosylation Factors↗

Properties of the interaction of Arf-like protein 2 with PDEdelta.

Arf-like proteins (Arl) share certain characteristic features with the Arf subfamily of Ras superfamily proteins, but their function is unknown. Here, we show by a variety of spectroscopic techniques that Arl2, unlike most other Ras-related proteins, has micromolar rather than picomolar affinity for nucleotides. As a consequence of low affinity, nucleotide dissociation rates are rather fast, arguing that it is not regulated by guanine nucleotide exchange factors. Arl2 is isolated as prey in a yeast double hybrid screen using phosphodiesterase 6delta (PDEdelta) as bait. This interaction is dependent on GTP, and the binding of PDEdelta substantially stabilizes GTP binding, increasing affinity and decreasing dissociation rates by a similar factor. Among all Arl proteins tested, PDEdelta only interacted with the closely related proteins Arl2 and Arl3, strongly suggesting that Arl2/3 are specific regulators of PDEdelta.

3',5'-Cyclic-GMP Phosphodiesterases↗

The TP53-ARF tumor suppressor pathway is frequently disrupted in large/cell anaplastic medulloblastoma.

We analyzed the TP53 and INK4A/ARF loci in 29 pediatric medulloblastomas. Mutually exclusive mutation in TP53, methylation of P14(ARF) or deletion of INK4A/ARF were identified in 21% (6/29) of tumors. Five of these alterations were detected in large cell/anaplastic medulloblastomas or tumors with significant anaplasia. Our data provide the first evidence that alterations within the TP53-ARF tumor suppressor pathway contribute to development of aggressive forms of medulloblastoma.

Anaplasia↗

Autophagy by ARF: a short story.

In this issue of Molecular Cell, Reef et al. (2006) describe a shortened unstable form of the ARF tumor suppressor protein that localizes within mitochondria, where it reduces membrane potential and triggers autophagy. Could this account for the Mdm2- and p53-independent tumor suppressive effects of ARF?

Animals↗

MDM2 and MDMX can interact differently with ARF and members of the p53 family.

Members of the p53 family of transcription factors have essential roles in tumor suppression and in development. MDM2 is an essential regulator of p53 that can inhibit the transcriptional activity of p53, shuttle p53 out of the nucleus, and target p53 for ubiquitination-mediated degradation. Little is known about the interaction and selectivity of different members of the p53 family (p53, p63, and p73) and the MDM2 family (MDM2 and MDMX). Here we show that the transcriptional activities of p53 and p73, but not that of p63, were inhibited by both MDM2 and MDMX. Consistent with these, we found that MDMX can physically interact with p53 and p73, but not with p63. Moreover, ectopically expressed MDM2 and MDMX could induce alterations in the subcellular localization of p73, but did not affect the subcellular localization of p53 and p63. Finally, we demonstrate that while ARF can interact with MDM2 and inhibit the regulation of p53 by MDM2, no interaction was found between ARF and MDMX. These data reveal that significant differences and selectivity exist between the regulation of different members of the p53 family by MDM2 and MDMX.

ADP-Ribosylation Factors↗

Regulation of exocytosis in adrenal chromaffin cells: focus on ARF and Rho GTPases.

Neurons and neuroendocrine cells release transmitters and hormones by exocytosis, a highly regulated process in which secretory vesicles or granules fuse with the plasma membrane to release their contents in response to a calcium trigger. Several stages have been recognized in exocytosis. After recruitment and docking at the plasma membrane, vesicles/granules enter a priming step, which is then followed by the fusion process. Cortical actin remodelling accompanies the exocytotic reaction, but the links between actin dynamics and trafficking events remain poorly understood. Here, we review the action of Rho and ADP-ribosylation factor (ARF) GTPases within the exocytotic pathway in adrenal chromaffin cells. Rho proteins are well known for their pivotal role in regulating the actin cytoskeleton. ARFs were originally identified as regulators of vesicle transport within cells. The possible interplay between these two families of GTPases and their downstream effectors provides novel insights into the mechanisms that govern exocytosis.

ADP-Ribosylation Factors↗

ARF is not required for apoptosis in Rb mutant mouse embryos.

The retinoblastoma (RB) tumor suppressor gene occupies central roles in cell cycle control and tumor suppression. Homozygous mutant (Rb(-/-)) embryos die at E13.5-E15.5, exhibiting extensive apoptosis and inappropriate S phase entry in the central and peripheral nervous systems, liver, and ocular lens. Mice simultaneously mutant for Rb and other genes can be generated to assess the requirement for these genes in cell cycle control and apoptosis. Using such analysis, E2f-1, E2f-3, p53, and Id2 have been identified as important regulators of cell cycle control and apoptosis in Rb(-/-) embryos. Because unrestrained E2F activity in the absence of Rb function contributes to p53-dependent apoptosis in many systems, we wished to identify genes linking deregulated E2F activity to p53 activation and subsequent apoptosis. As a transcriptional target of E2F-1, a regulator of p53, and an important mediator of apoptosis, ARF was a strong candidate for such a role, especially since it can be upregulated in the absence of Rb. From the analysis of Rb/ARF compound mutants we demonstrate that ARF is not an obligatory link between Rb inactivation and p53-dependent apoptosis.

ADP-Ribosylation Factors↗