Signal transduction cascades regulating fungal development and virulence.

Cellular differentiation, mating, and filamentous growth are regulated in many fungi by environmental and nutritional signals. For example, in response to nitrogen limitation, diploid cells of the yeast Saccharomyces cerevisiae undergo a dimorphic transition to filamentous growth referred to as pseudohyphal differentiation. Yeast filamentous growth is regulated, in part, by two conserved signal transduction cascades: a mitogen-activated protein kinase cascade and a G-protein regulated cyclic AMP signaling pathway. Related signaling cascades play an analogous role in regulating mating and virulence in the plant fungal pathogen Ustilago maydis and the human fungal pathogens Cryptococcus neoformans and Candida albicans. We review here studies on the signaling cascades that regulate development of these and other fungi. This analysis illustrates both how the model yeast S. cerevisiae can serve as a paradigm for signaling in other organisms and also how studies in other fungi provide insights into conserved signaling pathways that operate in many divergent organisms.

Cyclic AMP-dependent protein kinase controls virulence of the fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen that infects the human central nervous system. This pathogen elaborates two specialized virulence factors: the antioxidant melanin and an antiphagocytic immunosuppressive polysaccharide capsule. A signaling cascade controlling mating and virulence was identified. The PKA1 gene encoding the major cyclic AMP (cAMP)-dependent protein kinase catalytic subunit was identified and disrupted. pka1 mutant strains were sterile, failed to produce melanin or capsule, and were avirulent. The PKR1 gene encoding the protein kinase A (PKA) regulatory subunit was also identified and disrupted. pkr1 mutant strains overproduced capsule and were hypervirulent in animal models of cryptococcosis. pkr1 pka1 double mutant strains exhibited phenotypes similar to that of pka1 mutants, providing epistasis evidence that the Pka1 catalytic subunit functions downstream of the Pkr1 regulatory subunit. The PKA pathway was also shown to function downstream of the Galpha protein Gpa1 and to regulate cAMP production by feedback inhibition. These findings define a Galpha protein-cAMP-PKA signaling pathway regulating differentiation and virulence of a human fungal pathogen.

Signal transduction cascades regulating pseudohyphal differentiation of Saccharomyces cerevisiae.

In response to nitrogen limitation, diploid cells of the yeast Saccharomyces cerevisiae undergo a dimorphic transition to filamentous pseudohyphal growth. At least two signaling pathways regulate filamentation. One involves components of the MAP kinase cascade that also regulates mating of haploid cells. The second involves a nutrient-sensing G-protein-coupled receptor that signals via an unusual G(alpha) protein, cAMP and protein kinase A. Recent studies reveal crosstalk between these pathways during pseudohyphal growth. Related MAP kinase and cAMP pathways regulate filamentation and virulence of human and plant fungal pathogens, and represent novel targets for antifungal drug design.

The G protein-coupled receptor gpr1 is a nutrient sensor that regulates pseudohyphal differentiation in Saccharomyces cerevisiae.

Pseudohyphal differentiation in the budding yeast Saccharomyces cerevisiae is induced in diploid cells in response to nitrogen starvation and abundant fermentable carbon source. Filamentous growth requires at least two signaling pathways: the pheromone responsive MAP kinase cascade and the Gpa2p-cAMP-PKA signaling pathway. Recent studies have established a physical and functional link between the Galpha protein Gpa2 and the G protein-coupled receptor homolog Gpr1. We report here that the Gpr1 receptor is required for filamentous and haploid invasive growth and regulates expression of the cell surface flocculin Flo11. Epistasis analysis supports a model in which the Gpr1 receptor regulates pseudohyphal growth via the Gpa2p-cAMP-PKA pathway and independently of both the MAP kinase cascade and the PKA related kinase Sch9. Genetic and physiological studies indicate that the Gpr1 receptor is activated by glucose and other structurally related sugars. Because expression of the GPR1 gene is known to be induced by nitrogen starvation, the Gpr1 receptor may serve as a dual sensor of abundant carbon source (sugar ligand) and nitrogen starvation. In summary, our studies reveal a novel G protein-coupled receptor senses nutrients and regulates the dimorphic transition to filamentous growth via a Galpha protein-cAMP-PKA signal transduction cascade.

On chronological changes in the basic EEG rhythm in persons with Down syndrome - with special reference to slowing of alpha waves.

The authors tried to know specificity of aging in persons with Down syndrome (DS) from the aspect of electroencephalograph (EEG) frequency changes through the cross-sectional and longitudinal studies, in comparison with normal persons as well as those with mentally retardation except the Down syndrome (non-DS MR). Subjects for a cross-sectional study were 265 persons with DS, 242 with non-DS MR and 239 healthy persons, and subjects for a follow-up study were 28 persons with DS and 14 with non-DS MR, whose EEGs were recorded repeatedly once a year during 8 or 9 years. Resting EEGs from the frontal, central and occipital regions were examined through power spectrum. In the cross-sectional study, the number of subjects with DS who showed dominant component within 8 Hz band of the basic rhythm reached maximum in its appearance rate at 40-44 years of age in the occipital area, but this slowing progressed already at 30-34 years of age. While in non-DS MR, the number of subjects who showed dominant component at 8 Hz reached maximum at 45-49 years of age, and this slowing of the basic rhythm was not so clear as in DS. In the follow-up study for subjects with DS, although the lowering in EEG frequency to 8 Hz took place in various years of age individually, earlier distinct decrease of the frequency was commonly noticed. These earlier steep lowering of EEG frequency was discussed in relation to the senile signs and to the decline of brain function referring to Alzheimer disease.

Efficacy of Er:YAG laser irradiation in removing debris and smear layer on root canal walls.

The purpose of this study was to evaluate the efficacy of Er:YAG laser irradiation in removing debris and smear layer from prepared root canal walls. Thirty-six human extracted mandibular incisors teeth were divided into three groups. Group 1 (G1) was control specimens that were not lased. The teeth of group 2 (G2) and group 3 (G3) were irradiated by Er:YAG laser at different watt powers of 1 W and 2 W. The teeth were bisected and prepared for study in stereoscopic light microscope and SEM. Control specimens showed an amount of debris and heavy smear layer obscuring the dentinal tubules at all levels in the canals. The root canal walls irradiated by Er:YAG laser were free of debris, with an evaporated smear layer and open dentinal tubules. Statistical analyses showed significant differences (p < 0.01) in cleanliness smear layer between G1 and G2, and G1 and G3. However, there was no statistically significant difference between G2 and G3 in the cleanliness of the middle and apical one-third of the root canals. These results show Er:YAG laser is effective in removing debris and smear layer from root canal walls.

Effects of pulsed Nd:YAG laser irradiation on root canal wall dentin with different laser initiators.

The effects of pulsed Nd:YAG laser irradiation with different laser initiators on the permeability and ultrastructure of the root canal wall dentin were investigated in vitro. Forty extracted human single-rooted teeth were randomly assigned to four groups. Group 1 teeth were not lased as a control. Group 2 specimens received four 10-s duration laser exposures for a total exposure of 40 s/canal. In group 3 specimens, the root canals were painted with black ink and then lased by the same method as group 2 teeth. In group 4 specimens, root canals were treated with 38% Ag(NH3)2F and then lased by the same method as group 2 teeth. Laser parameters were set at 2 W, 20 pps. After being placed in 0.6% rhodamine B solution for 48 h, the teeth were sectioned for study by stereoscope and scanning electron microscopy. Statistical analysis showed there were significant differences (p < 0.05) in dentin permeability in the apical areas between groups 3 and 1, 4 and 1, and 4 and 2. Scanning electron microscopic examination showed that laser treatment alone had no obvious effects on the root canal wall. The root canal surfaces prepared for by laser irradiation with black ink or 38% Ag(NH3)2F revealed melting, smear layer evaporation, and open dentinal tubules. Black ink was more effective than 38% Ag(NH3)2F as a Nd:YAG laser initiator.

Effects of CO2 laser in treatment of cervical dentinal hypersensitivity.

The effectiveness of CO2 laser therapy in the reduction and elimination of dentinal hypersensitivity in vivo and its thermal effects on tooth surfaces in vitro were investigated. Twenty-three patients with 91 sensitive teeth participated in this study and were followed for 3 months. The parameters used with CO2 laser were 1 W in a continuous wave mode and irradiation time ranging from 5 to 10 s. Hypersensitivity was assessed by thermal stimulus (a blast of air from a dental syringe). Thermal effects were measured by thermography using 10 extracted human teeth. After laser treatment, all patients were immediately free from sensitive pain. Over 3 months, the CO2 laser treatment reduced dentinal hypersensitivity to air stimulus by 50%. All teeth remained vital with no adverse effects. Thermography revealed no temperature increase on irradiated tooth surfaces subjected to water coolant. These results show that the CO2 laser is useful in the treatment of cervical dentinal hypersensitivity without thermal damage to pulp.

Multiple optical stimulation to neuron using Si opto-neural probe with multiple optical waveguides and metal-cover for optogenetics.

We have developed a Si opt-neural probe with multiple waveguides and metal cover for highly accurate optical stimulation. This neural probe had 16 recording sites, three optical waveguides, and metal cover for suppressing light leakage. We evaluated electrochemical properties of the recording sites, and confirmed that the neural probe had suitable characteristics for neural recording. We also demonstrated the optical stimulation to the neurons expressing ChR2 using our probe. As a result, we succeeded multisite optical stimulation, and observed that no light leakage from the optical waveguides because of the metal cover. From in vivo experiments, we successfully recorded optically modulated local field potential using the fabricated Si neural probe with optical waveguides. Moreover, we applied current source density analysis to the recorded LFPs. As a result, we confirmed that light induced membrane current sink in locally stimulated area. Our Si opto-neural probe with multiple optical waveguides and metal-cover is one of the most versatile tools for optogenetics.

Age-Related Changes in Auditory and Cognitive Abilities in Elderly Persons with Hearing Aids Fitted at the Initial Stages of Hearing Loss.

In this study, we investigated the relation between the use of hearing aids at the initial stages of hearing loss and age-related changes in the auditory and cognitive abilities of elderly persons. 12 healthy elderly persons participated in an annual auditory and cognitive longitudinal examination for three years. According to their hearing level, they were divided into 3 subgroups - the normal hearing group, the hearing loss without hearing aids group, and the hearing loss with hearing aids group. All the subjects underwent 4 tests: pure-tone audiometry, syllable intelligibility test, dichotic listening test (DLT), and Wechsler Adult Intelligence Scale-Revised (WAIS-R) Short Forms. Comparison between the 3 groups revealed that the hearing loss without hearing aids group showed the lowest scores for the performance tasks, in contrast to the hearing level and intelligibility results. The other groups showed no significant difference in the WAIS-R subtests. This result indicates that prescription of a hearing aid during the early stages of hearing loss is related to the retention of cognitive abilities in such elderly people. However, there were no statistical significant correlations between the auditory and cognitive tasks.

Pleiotropic deficiencies of the laccase-derepressed mutant lah-1 are caused by constitutively increased expression of the cross-pathway control gene cpc-1 in Neurospora crassa.

In Neurospora crassa, expression of the laccase gene is induced by treatment with the protein synthesis inhibitor cycloheximide (CHX). This expression is mediated by CPC1, which acts as a general transcriptional activator when mycelia are treated with CHX or starved for any one of the amino acids. A laccase-derepressed mutant, lah-1, shows pleiotropic deficiencies in growth, hyphal morphology, CHX sensitivity, and production of protoperithecia. Moreover, in the lah-1 mutant, transcript levels of CHX-inducible genes, including lacc, tub-2, tef-1, and amino acid biosynthetic genes such as cpc-1, trp-3, and arg-12, are increased without exposure to CHX. All of the defects exhibited in the lah-1 mutant are suppressed by a mutation in the cpc-1 locus. These findings suggest that the cpc-1 mutation is epistatic to the lah-1 mutation and that the pleiotropic defects in the lah-1 mutant are attributable to constitutive expression of CPCI. These conclusions are supported by a developmental Northern blot analysis of the CHX-inducible genes. Based on these results, the lah-1 gene product appears to regulate expression of the cpc-1 gene negatively. Expression of the CHX-inducible genes was induced by CHX treatment in the lah-1 cpc-1 mutant, as well as in the cpc-1 mutant. This observation indicates that LAH1 is not a component of CHX-responsive pathway itself.

Isolation and characterization of mutants defective in production of laccase in Neurospora crassa.

A protein synthesis inhibitor, cycloheximide, induces excretion of laccase in Neurospora crassa. The lah-1 mutation results in excretion of a large amount of laccase even in the absence of cycloheximide. Ten mutations were induced that suppress derepressed excretion of laccase in the lah-1 mutant. Of these, seven second-site mutations were found to confer a laccase-noninducible phenotype, and were classified into two different complementation groups. Four mutations define a locus designated lni-1, found to be closely linked to ylo-1 on linkage group VI. The other three mutations were mapped to second locus, designated lni-2, that lies between nic-3 and thi-3 on linkage group VII. The lni-2 locus was shown to encode laccase by RFLP mapping of the DNA fragment encoding laccase and by transformation of the lni-2 mutant with plasmid pBL1 carrying the laccase gene (the locus encoding laccas is hereafter described as lacc). All lacc mutants examined (whether mutagen-induced or inactivated by repeat-induced point mutation) appeared to exhibit no phenotypic deficiency during both asexual and sexual cycles, suggesting that the laccase gene is dispensable in N. crassa. Northern analysis of total cellular RNA from the four lni-1 mutants demonstrated that the lni-1 mutations abolish increased transcription of the laccase gene under inducing conditions. Consequently, the lni-1 locus is inferred to encode a trans-acting positive regulator required for transcriptional activation of the laccase gene in response to cycloheximide. Possible functions of the lah-1 gene are also described.

A comparative study of the removal of smear layer by three endodontic irrigants and two types of laser.

AIM: The effects of three endodontic irrigants and two types of laser on a smear layer created by hand instrumentation were evaluated in vitro in the middle and apical thirds of root canals. METHODOLOGY: Sixty human mature extracted mandibular premolar teeth with a single root canal and a closed apex were distributed randomly into five groups of 12 teeth each. Whilst cleaning and shaping up to a size 60 master apical file with a step-back technique, the root canals were irrigated with 3 mL of 5.25% NaOCL and 3% H2O2, alternately, between each file size. Group 1 (G1) were control specimens that were irrigated with a final flush of 17% EDTA. The teeth in group 2 (G2) were irrigated with a final flush of 6% phosphoric acid, and group 3 (G3) with 6% citric acid. In the specimens of group 4 (G4) the root canals were irradiated with a carbon dioxide (CO2) laser, and specimens of group 5 (G5) were irradiated using an Er:YAG laser. The teeth were split longitudinally and prepared for examination by scanning electron microscopy. RESULTS: Control specimens (G1) showed clean root-canal walls with open dentinal tubules in the middle one-third, but in some specimens thick smear layer was observed in the apical one-third. Specimens irrigated with a final flush of 6% phosphoric acid (G2) or 6% citric acid (G3) were cleaner than with 17% EDTA, showing very clean root canal surfaces in the middle one-third but in the apical one-third the smear layer was not completely removed, especially at the openings of the dentinal tubules. The specimens irradiated with the CO2 laser (G4) showed clean root-canal walls with the smear layer absent, charred, melted, recrystallized and glazed in both middle and apical thirds. The root-canal walls of the specimens irradiated with the Er:YAG laser (G5) revealed an absent smear layer with open dentinal tubules in the middle and apical thirds. Statistical analysis showed no significant difference in the cleanliness of root-canal wall between G1 and G2, and G1 and G3. However, there were statistically significant differences (P < 0.01) between G1 and G4, and G1 and G5 in the cleanliness of the middle and apical one-thirds of the root canals. CONCLUSIONS: Irrigation with 17% EDTA, 6% phosphoric acid and 6% citric acid did not remove all the smear layer from the root-canal system. In addition, these acidic solutions demineralized the interbular dentine around tabular openings, which became enlarged. The CO2 laser was useful in removing and melting the smear layer on the instrumented root-canal walls and the Er:YAG laser was the most effective in removing the smear layer from the root-canal wall.

Comparative study about the removal of smear layer by three types of laser devices.

OBJECTIVE: The aim of this study was to analyze the effectiveness of three types of laser, argon, Nd:YAG, and Er:YAG, to remove the smear layer from the prepared root canal walls in vitro. METHODS: After cleaning and shaping by step-back preparation, 32 human extracted maxillary molar teeth were divided randomly into four groups. Root canals in group 1 (G1) were unlased and were irrigated by 17% EDTA. In group (G2), root canals were irradiated by argon laser at the parameters of 1 W, 50 mJ, and 5 Hz. In group 3 (G3) root canals received Nd:YAG laser irradiation at 2 W, 200 mJ and 20 Hz. Teeth in group 4 (G4) were irradiated by Er:YAG laser at the following parameters: 1 W, 100 mJ and 10 Hz. Then these teeth were bisected longitudinally, observed using SEM, and evaluated. RESULTS: The middle third of the teeth from G1 showed clean wall surfaces with open dentinal tubules. In G2, at the middle third, the smear layer was free and vaporized pulpal tissue remnants were observed. In G3, most of the specimens showed very clean walls with the smear layer evaporated, melted, fused, and recrystallized in both the middle and apical thirds. The walls of G4 revealed the evaporated smear layer and open dentinal tubules in the middle and apical thirds. Statistical analysis showed significant differences between G1 and G2, G1 and G3, and G1 and G4. CONCLUSIONS: These results show that the argon laser and Nd:YAG laser are useful to remove the smear layer and that the Er:YAG laser irradiation is the most effective to remove the smear layer on root canal walls.

Transcriptional activation of a cycloheximide-inducible gene encoding laccase is mediated by cpc-1, the cross-pathway control gene, in Neurospora crassa.

Expression of the laccase gene (lacc) of Neurospora crassa is transcriptionally inducible by the protein synthesis inhibitor cycloheximide. A lni-1 mutation, conferring the laccase non-inducible phenotype, was found to be a cpc-1 allele. Northern blots probed with plasmid pLA1, which carries the lacc gene revealed that the cpc-1 mutation abolishes the induced transcription of the lacc gene, indicating requirement of the cpc-1 gene for transcriptional activation of the lacc gene. In Northern blots probed with plasmid pAB1, which bears arg-2 a gene whose transcription is under the control of CPC1, the level of the arg-2 transcript was shown to increase several-fold in wild-type mycelia but remained low in cpc-1 mycelia, after treatment with cycloheximide. This suggests that inhibition of protein synthesis with cycloheximide, as well as amino acid limitation, elicits the CPC1-mediated cross-pathway control. Characterization of the lacc upstream region using a series of 5'-deletion plasmids led to the identification of a 170 bp DNA region required for the induced lacc expression. Sequence analysis of this DNA region demonstrated that it includes a 9 bp sequence with dyad symmetry, ATGAATCAT, which differs only by a central base pair from ATGA(C/G)TCAT, the recognition sequence characteristic of CPC1 and GCN4 binding sites. Possible mechanisms by which CPC1 mediates transcriptional activation of the lacc gene are discussed.

Effect of Nd:YAG laser irradiation for removal of intracanal debris and smear layer in extracted human teeth.

OBJECTIVE: The objective of this study was to evaluate the efficacy of Nd:YAG laser to remove debris and smear layer on the instrumented root canal walls in vitro. SUMMARY BACKGROUND DATA: There have been some contradictory reports about the efficacy of Nd:YAG laser to remove debris and smear layer. MATERIALS AND METHODS: Thirty-six extracted single mandibular incisor human teeth were used in this study. The teeth were sectioned at an enamel-cementum junction and the roots were instrumented up to # 50-K files size, then divided into three groups of 12 teeth. The first group (Group 1) was unlased as a control, and in the other two groups, root canals were irradiated by Nd:YAG laser at two different parameters; 1 W, 20 pps (Group 2) and 2 W, 20 pps (Group 3). After laser irradiation, the roots were bisected longitudinally, observed by scanning electron microscopy, and evaluated the condition of cleanliness on the root canal walls according to our criteria. RESULTS: In most of the specimens in Groups 1 and 2, debris and smear layer remained on the root canal wall surfaces covering the orifices of dentinal tubules. However, specimen in Group 3 showed very clean root canal walls with debris and smear layer evaporated, melted, fused, and recrystallized in most cases. Statistical analysis demonstrated a significant difference between Groups 1 and 3. CONCLUSIONS: These results suggest that Nd:YAG laser is useful to remove debris and smear layer and causes melting of internal structures on the instrumented root canal walls at the parameters of 2 W and 20 pps.

Formation of the mutagenic/carcinogenic imidazoquinoxaline-type heterocyclic amines through the unstable free radical Maillard intermediates and its inhibition by phenolic antioxidants.

Generation of the imidazoquinoxaline-type heterocyclic amines in the heated model system composed of glucose/glycine/creatinine in aqueous diethylene glycol was effectively prevented by phenolic antioxidants, butylated hydroxyanisole (BHA), propyl gallate (PG), sesamol, esculetin and epigallocatechin gallate (EGCG) in a dose-dependent manner. Generation of the mutagens in heated-and-dried bonito meat was effectively prevented on pretreatment with EGCG or green tea extract. Electron spin resonance (ESR) studies showed that the heated model mixture of glucose/glycine generated the unstable pyrazine cation radical, and its formation was inhibited by BHA, sesamol and EGCG. ESR-spin trapping studies using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and N-tert-butyl-alpha-phenylnitrone (PBN) showed that the heated model mixture of glucose/glycine or glucose/glycine/creatinine generated unstable carbon-centred radical(s), and their formation was effectively inhibited by BHA, sesamol and EGCG. It is likely that the unstable free radical Maillard intermediates played an important role in the formation of the imidazoquinoxaline-type heterocyclic amines, and the phenolic antioxidants effectively scavenged the radical species to prevent the mutagen formation.

Isolation and characterization of the krev-1 gene, a novel member of ras superfamily in Neurospora crassa: involvement in sexual cycle progression.

Genes belonging to the ras superfamily encode low-molecular-weight GTP/GDP-binding proteins that are highly conserved in wide variety of organisms. We used the polymerase chain reaction (PCR) to isolate a novel member of the ras superfamily from the filamentous fungus Neurospora crassa and obtained a mammalian Krev-1 homolog. We named the gene krev-1 and analyzed its structure and function. The krev-1 gene encodes a polypeptide of 225 amino acids, which is nearly 60% homologous to the mammalian Krev-1 p21. The krev-1 gene product (KREV1) is functionally analogous to mammalian Krev-1 p21 and Rsr1p/Bud1p, a Krev-1 homolog from the yeast Saccharomyces cerevisiae. GAL1-driven expression of KREV1 in a wild-type yeast strain resulted in a random budding pattern, as did its mammalian counterpart Krev-1 p21. We disrupted the krev-1 gene by RIP (repeat-induced point mutation), but the krev-1 disruptants showed no abnormalities. By in vitro mutagenesis, we constructed several mutant krev-1 genes (G21V, A68T, and D128A) which mimic constitutively active mutants of Ha-ras, and the krev-1 (K25N) mutant which is analogous to a dominant-negative mutant of Ha-ras. Each mutant gene was introduced into the wild-type strain and the phenotypes were analyzed. We could not observe any difference in vegetative growth between these transformants. When each strain was used as the female in mating tests, the development of perithecia from protoperithecia was inhibited in all cases. The results indicate that the krev-1 gene may be involved in sexual cycle progression.

Effect of Er:YAG laser treatment on the root canal walls of human teeth: an SEM study.

The purposes of the study were to observe the morphological changes on root canal walls after instrumentation and irrigation, and assess the efficacy of conventional cleansing procedures and the effectiveness of Er:YAG laser in removing debris and smear layer from the root canal walls. Thirty-six endodontically treated human mandibular incisor teeth with single root canals were bisected longitudinally and divided into three groups of 12 teeth. Group 1 (G1) was left unlased as a control. The teeth of group 2 (G2 and group 3 (G3) were irradiated by Er:YAG laser (laser parameters were set at 1 W, 100 mJ/pulse and 10 Hz) for 3 s and 5 s. The teeth were prepared for scanning electron microscope study. Control specimens showed debris and heavy smear layer obscuring the dentinal tubules at all levels in the canals. The root canal walls irradiated by Er:YAG laser were free of debris, with an evaporated smear layer and open dentinal tubules. These results suggested that Er:YAG laser irradiation had an efficient cleaning effect on the prepared root canal walls.

Effect of argon laser irradiation on instrumented root canal walls.

The objective of the study was to examine whether argon laser has a property to remove debris and smear layer from root canal walls. Twelve endodontically treated human maxillary molar teeth with three root canals were divided into two groups of six teeth. The first group was left unlased as a control; in the second group the root canals were irradiated by argon laser (laser parameters were set at 1 W and pulse duration and pulse frequency fixed at 0.05 s and 5 Hz). After the usual root canal preparation and lasing had been carried out, the teeth were decoronated, bisected longitudinally, observed with a scanning electron microscope and evaluated as to how clean the surfaces of root canal walls were. In most cases control teeth presented surfaces with debris covering the root canals, obscuring the dentinal tubules. Only 1 of 18 specimens was free of debris. In the lased group, root canal surfaces free of debris and vaporized pulpal tissue remnants were observed in 13 of 18 specimens. The results showed significant statistical differences between the control group and the lased groups (P < 0.001). These results suggested that argon laser irradiation has an efficient cleaning effect on instrumented root canal surfaces.