Analysis of the Differentiation of Kenyon Cell Subtypes Using Three Mushroom Body-Preferential Genes during Metamorphosis in the Honeybee (Apis mellifera L.).

The adult honeybee (Apis mellifera L.) mushroom bodies (MBs, a higher center in the insect brain) comprise four subtypes of intrinsic neurons: the class-I large-, middle-, and small-type Kenyon cells (lKCs, mKCs, and sKCs, respectively), and class-II KCs. Analysis of the differentiation of KC subtypes during metamorphosis is important for the better understanding of the roles of KC subtypes related to the honeybee behaviors. In the present study, aiming at identifying marker genes for KC subtypes, we used a cDNA microarray to comprehensively search for genes expressed in an MB-preferential manner in the honeybee brain. Among the 18 genes identified, we further analyzed three genes whose expression was enriched in the MBs: phospholipase C epsilon (PLCe), synaptotagmin 14 (Syt14), and discs large homolog 5 (dlg5). Quantitative reverse transcription-polymerase chain reaction analysis revealed that expression of PLCe, Syt14, and dlg5 was more enriched in the MBs than in the other brain regions by approximately 31-, 6.8-, and 5.6-fold, respectively. In situ hybridization revealed that expression of both Syt14 and dlg5 was enriched in the lKCs but not in the mKCs and sKCs, whereas expression of PLCe was similar in all KC subtypes (the entire MBs) in the honeybee brain, suggesting that Syt14 and dlg5, and PLCe are available as marker genes for the lKCs, and all KC subtypes, respectively. In situ hybridization revealed that expression of PLCe is already detectable in the class-II KCs at the larval fifth instar feeding stage, indicating that PLCe expression is a characteristic common to the larval and adult MBs. In contrast, expression of both Syt14 and dlg5 became detectable at the day three pupa, indicating that Syt14 and dlg5 expressions are characteristic to the late pupal and adult MBs and the lKC specific molecular characteristics are established during the late pupal stages.

Novel middle-type Kenyon cells in the honeybee brain revealed by area-preferential gene expression analysis.

The mushroom bodies (a higher center) of the honeybee (Apis mellifera L) brain were considered to comprise three types of intrinsic neurons, including large- and small-type Kenyon cells that have distinct gene expression profiles. Although previous neural activity mapping using the immediate early gene kakusei suggested that small-type Kenyon cells are mainly active in forager brains, the precise Kenyon cell types that are active in the forager brain remain to be elucidated. We searched for novel gene(s) that are expressed in an area-preferential manner in the honeybee brain. By identifying and analyzing expression of a gene that we termed mKast (middle-type Kenyon cell-preferential arrestin-related protein), we discovered novel 'middle-type Kenyon cells' that are sandwiched between large- and small-type Kenyon cells and have a gene expression profile almost complementary to those of large- and small-type Kenyon cells. Expression analysis of kakusei revealed that both small-type Kenyon cells and some middle-type Kenyon cells are active in the forager brains, suggesting their possible involvement in information processing during the foraging flight. mKast expression began after the differentiation of small- and large-type Kenyon cells during metamorphosis, suggesting that middle-type Kenyon cells differentiate by modifying some characteristics of large- and/or small-type Kenyon cells. Interestingly, CaMKII and mKast, marker genes for large- and middle-type Kenyon cells, respectively, were preferentially expressed in a distinct set of optic lobe (a visual center) neurons. Our findings suggested that it is not simply the Kenyon cell-preferential gene expression profiles, rather, a 'clustering' of neurons with similar gene expression profiles as particular Kenyon cell types that characterize the honeybee mushroom body structure.

In situ hybridization analysis of the expression of futsch, tau, and MESK2 homologues in the brain of the European honeybee (Apis mellifera L.).

BACKGROUND: The importance of visual sense in Hymenopteran social behavior is suggested by the existence of a Hymenopteran insect-specific neural circuit related to visual processing and the fact that worker honeybee brain changes morphologically according to its foraging experience. To analyze molecular and neural bases that underlie the visual abilities of the honeybees, we used a cDNA microarray to search for gene(s) expressed in a neural cell-type preferential manner in a visual center of the honeybee brain, the optic lobes (OLs). METHODOLOGY/PRINCIPAL FINDINGS: Expression analysis of candidate genes using in situ hybridization revealed two genes expressed in a neural cell-type preferential manner in the OLs. One is a homologue of Drosophila futsch, which encodes a microtubule-associated protein and is preferentially expressed in the monopolar cells in the lamina of the OLs. The gene for another microtubule-associated protein, tau, which functionally overlaps with futsch, was also preferentially expressed in the monopolar cells, strongly suggesting the functional importance of these two microtubule-associated proteins in monopolar cells. The other gene encoded a homologue of Misexpression Suppressor of Dominant-negative Kinase Suppressor of Ras 2 (MESK2), which might activate Ras/MAPK-signaling in Drosophila. MESK2 was expressed preferentially in a subclass of neurons located in the ventral region between the lamina and medulla neuropil in the OLs, suggesting that this subclass is a novel OL neuron type characterized by MESK2-expression. These three genes exhibited similar expression patterns in the worker, drone, and queen brains, suggesting that they function similarly irrespective of the honeybee sex or caste. CONCLUSIONS: Here we identified genes that are expressed in a monopolar cell (Amfutsch and Amtau) or ventral medulla-preferential manner (AmMESK2) in insect OLs. These genes may aid in visualizing neurites of monopolar cells and ventral medulla cells, as well as in analyzing the function of these neurons.

Effect of air-drying and solvent evaporation on the strength of HEMA-rich versus HEMA-free one-step adhesives.

OBJECTIVES: The objectives of this study were (1) to clarify the relationship between the duration of air-drying of one-step self-etch adhesives (1-SEAs) and the evaporation degree (ED) of solvents, and (2) to evaluate the effect of ED on the ultimate micro-tensile strength (microTS) of the adhesives. METHODS: The ED of one HEMA-rich 1-SEA, Clearfil S3 Bond (Kuraray), and two HEMA-free 1-SEAs, iBond (Heraeus-Kulzer) and G-Bond (GC), was determined without air-drying and after, respectively, 5- and 10-s air-drying using a gravimetric method. Next, the microTS of the adhesives at ED equivalent to 0-, 5- and 10-s air-drying was measured. RESULTS: The ED increased with extension of air-drying time. Among the adhesives tested, iBond showed the largest ED, followed by G-Bond and Clearfil S3 Bond in this order. A longer air-drying time for 10-s resulted in a statistically significantly higher microTS for the HEMA-rich Clearfil S3 Bond. The microTS of the latter was higher than that of the other two HEMA-free adhesives for each air-drying time. SIGNIFICANCE: Air-drying of 1-SEAs had a significant effect on the degree of solvent evaporation (ED) and also on the mechanical properties (microTS) of the 1-SEAs upon setting. It is therefore beneficial to remove solvents of the 1-SEAs as much as possible by thorough, strong air-drying in order to achieve a strong adhesive layer at the interface.

Microrotary fatigue resistance of a HEMA-free all-in-one adhesive bonded to dentin.

PURPOSE: To determine the effect of thorough air drying on the static and dynamic bonding effectiveness of a HEMA-free all-in-one adhesive bonded to Class-1 cavity-bottom dentin. MATERIALS AND METHODS: G-Bond (GC, Japan) was applied to standard occlusal Class I cavities following either a gentle (1 bar) or strong (4 bar) air-drying technique. After restoring the cavity using a composite resin (Gradia Anterior, GC), microspecimens were prepared that were loaded until failure or inserted in a microrotary fatigue testing device. RESULTS: Strong air drying had no effect on the static (39.1 vs. 38.4 MPa) or the dynamic (19.0 vs. 18.6 MPa) strength of the interface complex. CONCLUSION: Strong air drying did not improve the static or the dynamic bonding effectiveness, although a very thorough (4 bar) and long air-drying procedure was employed. SEM analysis revealed that even when the adhesive was strongly air dried, droplets originating from phase separations could still be observed, albeit to a reduced extent. Ineffective drying and pooling of the adhesive in a narrow cavity diminished the effect of strong air drying of the all-in-one adhesive.

Immobilization of simulated reducing agent at the surface of SiO2 fillers in dental composite resins.

To reduce the leachability of reducing agents from composite resins, immobilization of a simulated reducing agent at the surface of SiO2 fillers was examined. SiO2 plates were immersed in 2% 3-aminopropyltriethoxy silane/ethanol solution, and then immersed in dimethyl sulfoxide with 0.25 wt% 4-dimethyl amino benzoic acid (DMABA), 2.0 wt% 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, and 0.5 wt% N-hydroxysuccinimide. Wide-scan spectrum of X-ray photoelectron spectroscopy did not detect carbon contamination. However, narrow scan detected an O=C-N peak at 399.8 eV, suggesting that DMABA could be immobilized on silane-coupled SiO2 plates. Further, surface plasmon resonance analysis indicated the adsorption of MMA at the surface of reducing agent-immobilized plate.

Effect of water storage on the bonding effectiveness of 6 adhesives to Class I cavity dentin.

Adhesive-dentin interfaces degrade with time. This study determined the effect water storage may have on the bonding effectiveness of adhesives to occlusal Class I cavity-bottom dentin. Six adhesives, all representing contemporary classes of adhesives, were applied: a 3-step (OptiBond FL, Kerr) and 2-step (Scotchbond 1*, 3M ESPE) etch-and-rinse adhesive, a 2-step (Clearfil SE, Kuraray) and 1-step (Adper prompt, 3M ESPE) self-etch adhesive and a 2-step (FujiBond LC, GC) and 1-step (Reactmer, Shofu) resin-modified glass-ionomer adhesive. Bonding effectiveness was assessed by microtensile bond strength testing (MTBS) and electron microscopy (Feg-SEM and TEM). The MTBS was determined after 1 day and 1 year water storage of the entire restored cavity (indirect exposure of the adhesive-dentin interface to water) and prepared microTBS-beams (direct exposure of the adhesive-dentin interface to water). The hypotheses tested were: (1) resin-dentin bonds formed at the bottom of Class I cavities resist 1-year water storage and (2) an adjacent composite-enamel bond protects the composite-dentin bond against degradation. Non-parametric Kruskal-Wallis analysis statistically analyzed the microTBSs. The first hypothesis was rejected, as only the microTBS of OptiBond FL and Clearfil SE did not significantly decrease after 1-year direct and/or indirect water storage. The second hypothesis was corroborated, as the bonding effectiveness of most simplified adhesives (Scotchbond 1, Adper Prompt, FujiBond LC and Reactmer) approached 0 (because of the frequent pre-testing failures) after 1-year direct water exposure. The second hypothesis concluded that the 3-step etch-and-rinse adhesive must still be regarded the "gold standard." Though microTBS decreased significantly, Clearfil SE, as a 2-step self-etch adhesive, was the only simplified adhesive to perform reliably after 1-year direct water exposure.

Differential expression of HR38 in the mushroom bodies of the honeybee brain depends on the caste and division of labor.

We used a cDNA microarray to identify genes expressed in a caste (worker)- and division of labor (nurse bees or foragers)-dependent manner in the honeybee brain. Among the identified genes, one encoded a putative orphan receptor (HR38) homologue that mediates ecdysteroid-signaling. Real-time reverse transcription-polymerase chain reaction indicated that expression of this gene is higher in forager brains, as compared to nurse bees and queens. In the forager brain, expression was concentrated in a subset of the mushroom body neurons, suggesting that ecdysteroid-signaling in the mushroom bodies might be involved in the division of labor of the workers.

Analysis of chemical interaction of 4-MET with hydroxyapatite using XPS.

Each dental adhesive contains a specific functional monomer that determines its actual adhesive performance to tooth tissue. 4-methacryloxyethyl trimellitic acid (4-MET) is well-known as one of the functional monomers mostly available and consequently widely used in commercial adhesives. We therefore characterized the chemical interaction of 4-MET with hydroxyapatite (HAp) using X-ray Photoelectron Spectroscopy (XPS). XPS revealed that the peak representing -COO- of 4-MET shifted to a lower binding energy, when 4-MET was adsorbed onto HAp. Deconvolution of this shifted peak disclosed two components with a peak representing unreacted carboxyl groups and ester groups, and a peak suggesting chemical bonding of other carboxyl groups to Ca of HAp. XPS spectra of HAp treated with 4-MET also disclosed the surface to be enriched in calcium and decreased in phosphorus, indicating that phosphorus was extracted at a relatively higher rate than calcium. It can thus be concluded that true chemical bonding of 4-MET with calcium present in HAp occurred, as it was proven using XPS.

Cytochrome P450 CYP307A1/Spook: a regulator for ecdysone synthesis in insects.

The prothoracic gland (PG) has essential roles in synthesizing and secreting a steroid hormone called ecdysone that is critical for molting and metamorphosis of insects. However, little is known about the genes controlling ecdysteroidogenesis in the PG. To identify genes functioning in the PG of the silkworm, Bombyx mori, we used differential display PCR and focused on a cytochrome P450 gene designated Cyp307a1. Its expression level positively correlates with a change in the hemolymph ecdysteroid titer. In addition, Drosophila Cyp307a1 is encoded in the spook locus, one of the Halloween mutant family members showing a low ecdysone titer in vivo, suggesting that Cyp307a1 is involved in ecdysone synthesis. While Drosophila Cyp307a1 is expressed in the early embryos and adult ovaries, the expression is not observed in the PGs of embryos or third instar larvae. These results suggest a difference in the ecdysone synthesis pathways during larval development in these insects.

Effect of evaporation of primer components on ultimate tensile strengths of primer-adhesive mixture.

OBJECTIVE: The purpose of the present study was to evaluate the effect of evaporation of primer components on the ultimate tensile strength (UTS) of primer-adhesive mixtures. METHODS: The UTSs of 1:3 by weight primer-adhesive mixtures and of the pure adhesives (controls) were measured after 0-, 2-, 5-min and 24-h storage in a 37 degrees C oven for the three-step etch&rinse adhesive OptiBond FL (Kerr) and the two-step self-etch adhesive Clearfil SE (Kuraray). RESULTS: Evaporation of primer components increased with time for both adhesives investigated. OptiBond FL showed a higher evaporation of primer components than Clearfil SE at the 2-, 5-min and 24-h storage time. As compared to the pure adhesive, the UTS of the primer-adhesive mixture was lower after 0- and 2-min storage for OptiBond FL and after 0-, 2- and 5-min storage for Clearfil SE. The UTS of OptiBond FL was higher than that of Clearfil SE at the 0- and 2-min storage time, though there was no difference at the 5-min and 24-h storage time. SIGNIFICANCE: The degree of evaporation of primer components depended largely on the primer solvents employed. The significant decrease in UTS of adhesive resin when mixed with primer must be attributed to incomplete evaporation of primer components. Complete evaporation of solvents is hard to achieve, even by thorough air drying.

Is 2.5 mEq/L the optimal calcium concentration of dialysate in the use of sevelamer hydrochloride? A study of the dialysate calcium concentration recommended by K/DOQI guidelines.

We tested the effect of three different dialysate calcium concentrations on calcium-phosphorus metabolism during the use of sevelamer hydrochloride. After a calcium-containing phosphate binder was switched to sevelamer, the serum calcium, phosphorus, and intact parathyroid hormone levels and the markers of bone turnover were measured in the patients whose dialysate calcium concentrations were 2.5, 2.75, and 3.0 mEq/L. As a result, in the 2.75-mEq/L group, the serum calcium concentrations decreased and the intact parathyroid hormone level increased significantly. In the 2.5-mEq/L group, transient hypocalcemia occurred and the levels of both bone-alkaline phosphatase and osteocalcin increased. In the 3.0-mEq/L group, the serum calcium concentrations did not change significantly and only bone-alkaline phosphatase increased. If a calcium-containing phosphate binder is completely switched to sevelamer, dialysis using a dialysate calcium concentration below 3.0 mEq/L may result in hypocalcemia and acceleration of bone turnover.

Effect of fracture strength of primer-adhesive mixture on bonding effectiveness.

OBJECTIVE: The purpose of this study was to measure the micro-tensile bond strength (microTBS) of two adhesives to dentin and to correlate this microTBS with the micro-tensile fracture strength (microTFS) of primer-adhesive mixtures of the two adhesives. METHODS: The three-step etch&rinse adhesive OptiBond FL (Kerr) and the two-step self-etch adhesive Clearfil SE Bond (Kuraray) were used to bond Filtek Z100 (3M ESPE) to twenty mid-coronal dentin surfaces. The microTBS was determined at 1 and 24 h after light-curing. In addition, the microTFS of primer-adhesive mixtures (primer:adhesive=1:3 by weight) and of the pure adhesives (controls) were measured after the same time intervals. RESULTS: The microTBS to dentin of OptiBond FL at 24 h was significantly higher than at 1 h and than the microTBS of Clearfil SE Bond at 24 h. The mean microTFS of the primer-adhesive mixtures was always lower than the microTFS of the respective adhesives. The highest reduction in microTFS due to mixing primer with the adhesive resin was measured for Clearfil SE Bond. Both the microTFS of the primer-adhesive mixtures and the pure adhesives of OptiBond FL and Clearfil SE Bond increased with time. SIGNIFICANCE: The early 1 h microTBS to dentin was lower than the 24 h microTBS for OptiBond FL, but not for Clearfil SE Bond. Differences in polymerization efficiency as well as in the resulting interfacial strength may explain such varying early bond strengths to dentin.

Effect of cavity configuration and aging on the bonding effectiveness of six adhesives to dentin.

OBJECTIVES: The purpose of this study was to determine the effect polymerization contraction stress may have on bond durability. METHODS: Bonding effectiveness was assessed by micro-tensile bond strength testing (muTBS) and electron microscopy. The muTBS to flat dentin surfaces and in standardized cavities was determined (this after 1 day as well as 1 year water storage). Six adhesives representing all current classes were applied: two etch-and-rinse (OptiBond FL, Kerr; Scotchbond 1, 3M ESPE), two self-etch (Clearfil SE Bond, Kuraray; Adper Prompt, 3M ESPE) and two glass-ionomer (Fuji Bond LC, GC; Reactmer, Shofu) adhesives. RESULTS: The conventional 3-step etch-and-rinse adhesive OptiBond FL bonded most effectively to dentin, and appeared insensitive to polymerization shrinkage stress and water degradation. The 2-step self-etch adhesive Clearfil SE Bond most closely approached this superior bonding effectiveness and only slightly lost bond strength after 1-year water exposure. The 2-step etch-and-rinse adhesive Scotchbond 1 and the 'strong' 1-step self-etch adhesive Adper Prompt appeared very sensitive to cavity configuration and water-aging effects. The 2-step resin-modified glass-ionomer adhesive Fuji Bond LC only suffered from shrinkage stress, but not from 1-year water-exposure. Remarkable also is the apparent repairability of the 'mild' 1-step glass-ionomer adhesive Reactmer when stored for 1 year in water, in spite of the very low 1-day muTBS. SIGNIFICANCE: Simplified bonding procedures do not necessarily imply improved bonding performance, especially in the long term.

[A case of sarcoidosis overlapping with rheumatoid arthritis].

We report the case of a 54-year-old woman who presented on May 28, 2001 with sarcoidosis overlapping with rheumatoid arthritis. She had experienced morning stiffness 2 years previously and was diagnosed as having rheumatoid arthritis. She had been treated with bucillamine and loxoprofen for 3 months. In October 2000, she developed proteinurea. The patient discontinued treatment with bucillamine and loxoprofen. Proteinurea persisted, and the patient's renal function declined. On admission, subcutaneous nodules were palpable in the patient's legs. The patient's serum creatinine and calcium levels were 2.49 mg/dl and 11.6 mg/dl, respectively. Intact-PTH was suppressed, and PTHrP was not elevated. Despite the presence of hypercalcemia, the patient's serum 1 alpha 25(OH)2D3 was not suppressed. Serum ACE and lysozyme levels were elevated beyond the normal ranges. A renal biopsy was performed, and non-caseous epithelioid granuloma was found in the renal interstitium. Based on the histological findings, the patient was diagnosed as having sarcoidosis. Following treatment with prednisolone, the patient's serum calcium levels returned to normal and her renal function improved.

Identification of genes expressed preferentially in the honeybee mushroom bodies by combination of differential display and cDNA microarray.

To clarify the molecular basis underlying the neural function of the honeybee mushroom bodies (MBs), we identified three genes preferentially expressed in MB using cDNA microarrays containing 480 differential display-positive candidate cDNAs expressed locally or differentially, dependent on caste/aggressive behavior in the honeybee brain. One of the cDNAs encodes a putative type I inositol 1,4,5-trisphosphate (IP(3)) 5-phosphatase and was expressed preferentially in one of two types of intrinsic MB neurons, the large-type Kenyon cells, suggesting that IP(3)-mediated Ca(2+) signaling is enhanced in these neurons.

Effect of presilanization filler decontamination on aesthetics and degradation resistance of resin composites.

Filler-matrix coupling determines, to a large extent, the mechanical strength and clinical longevity of dental composites. The aim of this study was to examine how far a methodology to decontaminate filler prior to silanization may improve aesthetic performance in addition to physico-mechanical properties such as degradation resistance. It was reported that filler particles are surrounded and wrapped by a film that consists of multiple layers of silane molecules. X-ray photoelectron spectroscopy, however, revealed that silanization of filler particles largely depended upon siloxane bridge (Si-O-Si) formation between the silica surface and the silane molecule rather than on intermolecular bonding between adjacent silane molecules. In this study, we showed that filler decontamination resulted in a higher translucency, thereby providing a better aesthetic potential. In addition, experimental composites produced following presilanization decontamination of filler revealed a higher Vickers hardness value and a diametral tensile strength that was resistant to degradation by thermo-cycling.